t4 dna ligase  (New England Biolabs)


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  • 99
    Name:
    T4 DNA Ligase
    Description:
    T4 DNA Ligase 100 000 units
    Catalog Number:
    M0202L
    Price:
    256
    Size:
    100 000 units
    Category:
    DNA Ligases
    Score:
    85
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    Structured Review

    New England Biolabs t4 dna ligase
    T4 DNA Ligase
    T4 DNA Ligase 100 000 units
    https://www.bioz.com/result/t4 dna ligase/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase - by Bioz Stars, 2019-12
    99/100 stars

    Images

    1) Product Images from ""

    Article Title:

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.284992

    Reaction of T4 DNA ligase with substrate 1 ( A ) and adenylylated substrate 1A ( B ) under single turnover conditions.  Each reaction was run with 500 n m  ligase and 100 n m  substrate in the standard ATP-free assay buffer. Ligase that was  > 95% adenylylated was used for  A , and
    Figure Legend Snippet: Reaction of T4 DNA ligase with substrate 1 ( A ) and adenylylated substrate 1A ( B ) under single turnover conditions. Each reaction was run with 500 n m ligase and 100 n m substrate in the standard ATP-free assay buffer. Ligase that was > 95% adenylylated was used for A , and

    Techniques Used:

    Pre-steady state reactions of 30 n m  (♦) and 50 n m  (■) T4 DNA ligase with 100 n m  substrate 1.  Reactions were run in the standard assay buffer. Each time point represents the average of three experiments, and the  error bars  represent one S.D. The  dashed lines  represent fits by simulation using the chemical rates determined from single turnover reaction of substrate  1 , literature values for Step 1 rates, and diffusion-limited binding of DNA and allowing the rate of product release ( k off ) and the amplitude ( a ) to vary. The best fit was obtained with  a  = 0.51 and  k off  = 0.58 s −1 .
    Figure Legend Snippet: Pre-steady state reactions of 30 n m (♦) and 50 n m (■) T4 DNA ligase with 100 n m substrate 1. Reactions were run in the standard assay buffer. Each time point represents the average of three experiments, and the error bars represent one S.D. The dashed lines represent fits by simulation using the chemical rates determined from single turnover reaction of substrate 1 , literature values for Step 1 rates, and diffusion-limited binding of DNA and allowing the rate of product release ( k off ) and the amplitude ( a ) to vary. The best fit was obtained with a = 0.51 and k off = 0.58 s −1 .

    Techniques Used: Diffusion-based Assay, Binding Assay

    Determination of  k cat  and  k cat / K m  for T4 DNA ligase and nicked substrates.  Shown is reaction of 1 n m  T4 DNA ligase with 1 n m  (○), 2 n m  (*), 5 n m  (×), 10 n m  (△), 20 n m  (♢), and 50 n m  (□) substrate  1  in standard assay buffer at 16 °C ( A ) and 1 n m  T4 DNA ligase (
    Figure Legend Snippet: Determination of k cat and k cat / K m for T4 DNA ligase and nicked substrates. Shown is reaction of 1 n m T4 DNA ligase with 1 n m (○), 2 n m (*), 5 n m (×), 10 n m (△), 20 n m (♢), and 50 n m (□) substrate 1 in standard assay buffer at 16 °C ( A ) and 1 n m T4 DNA ligase (

    Techniques Used:

    2) Product Images from ""

    Article Title:

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.284992

    Reaction of T4 DNA ligase with substrate 1 ( A ) and adenylylated substrate 1A ( B ) under single turnover conditions.  Each reaction was run with 500 n m  ligase and 100 n m  substrate in the standard ATP-free assay buffer. Ligase that was  > 95% adenylylated was used for  A , and
    Figure Legend Snippet: Reaction of T4 DNA ligase with substrate 1 ( A ) and adenylylated substrate 1A ( B ) under single turnover conditions. Each reaction was run with 500 n m ligase and 100 n m substrate in the standard ATP-free assay buffer. Ligase that was > 95% adenylylated was used for A , and

    Techniques Used:

    Pre-steady state reactions of 30 n m  (♦) and 50 n m  (■) T4 DNA ligase with 100 n m  substrate 1.  Reactions were run in the standard assay buffer. Each time point represents the average of three experiments, and the  error bars  represent one S.D. The  dashed lines  represent fits by simulation using the chemical rates determined from single turnover reaction of substrate  1 , literature values for Step 1 rates, and diffusion-limited binding of DNA and allowing the rate of product release ( k off ) and the amplitude ( a ) to vary. The best fit was obtained with  a  = 0.51 and  k off  = 0.58 s −1 .
    Figure Legend Snippet: Pre-steady state reactions of 30 n m (♦) and 50 n m (■) T4 DNA ligase with 100 n m substrate 1. Reactions were run in the standard assay buffer. Each time point represents the average of three experiments, and the error bars represent one S.D. The dashed lines represent fits by simulation using the chemical rates determined from single turnover reaction of substrate 1 , literature values for Step 1 rates, and diffusion-limited binding of DNA and allowing the rate of product release ( k off ) and the amplitude ( a ) to vary. The best fit was obtained with a = 0.51 and k off = 0.58 s −1 .

    Techniques Used: Diffusion-based Assay, Binding Assay

    Determination of  k cat  and  k cat / K m  for T4 DNA ligase and nicked substrates.  Shown is reaction of 1 n m  T4 DNA ligase with 1 n m  (○), 2 n m  (*), 5 n m  (×), 10 n m  (△), 20 n m  (♢), and 50 n m  (□) substrate  1  in standard assay buffer at 16 °C ( A ) and 1 n m  T4 DNA ligase (
    Figure Legend Snippet: Determination of k cat and k cat / K m for T4 DNA ligase and nicked substrates. Shown is reaction of 1 n m T4 DNA ligase with 1 n m (○), 2 n m (*), 5 n m (×), 10 n m (△), 20 n m (♢), and 50 n m (□) substrate 1 in standard assay buffer at 16 °C ( A ) and 1 n m T4 DNA ligase (

    Techniques Used:

    3) Product Images from ""

    Article Title:

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.284992

    Reaction of T4 DNA ligase with substrate 1 ( A ) and adenylylated substrate 1A ( B ) under single turnover conditions.  Each reaction was run with 500 n m  ligase and 100 n m  substrate in the standard ATP-free assay buffer. Ligase that was  > 95% adenylylated was used for  A , and
    Figure Legend Snippet: Reaction of T4 DNA ligase with substrate 1 ( A ) and adenylylated substrate 1A ( B ) under single turnover conditions. Each reaction was run with 500 n m ligase and 100 n m substrate in the standard ATP-free assay buffer. Ligase that was > 95% adenylylated was used for A , and

    Techniques Used:

    Pre-steady state reactions of 30 n m  (♦) and 50 n m  (■) T4 DNA ligase with 100 n m  substrate 1.  Reactions were run in the standard assay buffer. Each time point represents the average of three experiments, and the  error bars  represent one S.D. The  dashed lines  represent fits by simulation using the chemical rates determined from single turnover reaction of substrate  1 , literature values for Step 1 rates, and diffusion-limited binding of DNA and allowing the rate of product release ( k off ) and the amplitude ( a ) to vary. The best fit was obtained with  a  = 0.51 and  k off  = 0.58 s −1 .
    Figure Legend Snippet: Pre-steady state reactions of 30 n m (♦) and 50 n m (■) T4 DNA ligase with 100 n m substrate 1. Reactions were run in the standard assay buffer. Each time point represents the average of three experiments, and the error bars represent one S.D. The dashed lines represent fits by simulation using the chemical rates determined from single turnover reaction of substrate 1 , literature values for Step 1 rates, and diffusion-limited binding of DNA and allowing the rate of product release ( k off ) and the amplitude ( a ) to vary. The best fit was obtained with a = 0.51 and k off = 0.58 s −1 .

    Techniques Used: Diffusion-based Assay, Binding Assay

    Determination of  k cat  and  k cat / K m  for T4 DNA ligase and nicked substrates.  Shown is reaction of 1 n m  T4 DNA ligase with 1 n m  (○), 2 n m  (*), 5 n m  (×), 10 n m  (△), 20 n m  (♢), and 50 n m  (□) substrate  1  in standard assay buffer at 16 °C ( A ) and 1 n m  T4 DNA ligase (
    Figure Legend Snippet: Determination of k cat and k cat / K m for T4 DNA ligase and nicked substrates. Shown is reaction of 1 n m T4 DNA ligase with 1 n m (○), 2 n m (*), 5 n m (×), 10 n m (△), 20 n m (♢), and 50 n m (□) substrate 1 in standard assay buffer at 16 °C ( A ) and 1 n m T4 DNA ligase (

    Techniques Used:

    4) Product Images from "Efficient assembly of very short oligonucleotides using T4 DNA Ligase"

    Article Title: Efficient assembly of very short oligonucleotides using T4 DNA Ligase

    Journal: BMC Research Notes

    doi: 10.1186/1756-0500-3-291

    Enhancement of T4 DNA ligase activity by supplemental oligonucleotides.  (a) Unsuccessful 4-bp duplex reactions could be salvaged by utilizing a supplementary oligonucleotide, designed to complement the first oligonucleotide-dsDNA duplex but is unphosphorylated to prevent ligation of itself. Two hour ligation of the 4-bp reaction at 16°C supplemented with 3.33 μM of the hexamer, shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆).  (b)  Ligation reaction of an octamer supplemented with a second octamer in which one is used for ligation and the other is used to extend the duplex. A two hour ligation at 16°C of serial concentrations of the octamer with 3.33 μM of the supplementary octamer shows significant ligation (■) compared to reactions without the supplemental octamer (◆).  (c)  Unsuccessful 3-bp duplex reactions could be salvaged by utilizing a supplementary hexamer that hybridized at all six positions. A two hour ligation of the 3-bp reaction at 16°C with 3.33 μM supplementary hexamer shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆).  (d)  Ligation using a hexamer pair at 4°C for 16 hours shows limited improvement (■) compared to the unsupplemented (◆) control.
    Figure Legend Snippet: Enhancement of T4 DNA ligase activity by supplemental oligonucleotides. (a) Unsuccessful 4-bp duplex reactions could be salvaged by utilizing a supplementary oligonucleotide, designed to complement the first oligonucleotide-dsDNA duplex but is unphosphorylated to prevent ligation of itself. Two hour ligation of the 4-bp reaction at 16°C supplemented with 3.33 μM of the hexamer, shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (b) Ligation reaction of an octamer supplemented with a second octamer in which one is used for ligation and the other is used to extend the duplex. A two hour ligation at 16°C of serial concentrations of the octamer with 3.33 μM of the supplementary octamer shows significant ligation (■) compared to reactions without the supplemental octamer (◆). (c) Unsuccessful 3-bp duplex reactions could be salvaged by utilizing a supplementary hexamer that hybridized at all six positions. A two hour ligation of the 3-bp reaction at 16°C with 3.33 μM supplementary hexamer shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (d) Ligation using a hexamer pair at 4°C for 16 hours shows limited improvement (■) compared to the unsupplemented (◆) control.

    Techniques Used: Activity Assay, Ligation

    Evaluation of minimal oligonucleotide substrate requirements for T4 DNA ligase.  (a) Schematic diagram of an immobilized DNA strand used in ligation assays and DNA construction. M-270 Dynabeads (Invitrogen) are attached through a streptavidin-biotin linkage to the 5' end of a double stranded DNA. The free end is designed with a variable 5' overhang, complementary to labeled oligonucleotides used in ligation. An additional BbsI restriction site and a forward primer site are included in the case of DNA construction.  (b)  Increasing concentrations of 5'-phosphorylated, 3'-fluorescently labeled oligonucleotide are ligated to 5 pmoles of immobilized dsDNA with a complementary overhang. Reactions were performed for one hour at 16°C and washed with TE to remove unligated substrate. Successful ligation kinetics are observed at the 5-bp duplex length (▲), but no significant ligation occurs at lengths of 4-bp (■) or 3-bp (◆).
    Figure Legend Snippet: Evaluation of minimal oligonucleotide substrate requirements for T4 DNA ligase. (a) Schematic diagram of an immobilized DNA strand used in ligation assays and DNA construction. M-270 Dynabeads (Invitrogen) are attached through a streptavidin-biotin linkage to the 5' end of a double stranded DNA. The free end is designed with a variable 5' overhang, complementary to labeled oligonucleotides used in ligation. An additional BbsI restriction site and a forward primer site are included in the case of DNA construction. (b) Increasing concentrations of 5'-phosphorylated, 3'-fluorescently labeled oligonucleotide are ligated to 5 pmoles of immobilized dsDNA with a complementary overhang. Reactions were performed for one hour at 16°C and washed with TE to remove unligated substrate. Successful ligation kinetics are observed at the 5-bp duplex length (▲), but no significant ligation occurs at lengths of 4-bp (■) or 3-bp (◆).

    Techniques Used: Ligation, Labeling

    5) Product Images from ""

    Article Title:

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.284992

    Reaction of T4 DNA ligase with substrate 1 ( A ) and adenylylated substrate 1A ( B ) under single turnover conditions.  Each reaction was run with 500 n m  ligase and 100 n m  substrate in the standard ATP-free assay buffer. Ligase that was  > 95% adenylylated was used for  A , and
    Figure Legend Snippet: Reaction of T4 DNA ligase with substrate 1 ( A ) and adenylylated substrate 1A ( B ) under single turnover conditions. Each reaction was run with 500 n m ligase and 100 n m substrate in the standard ATP-free assay buffer. Ligase that was > 95% adenylylated was used for A , and

    Techniques Used:

    Pre-steady state reactions of 30 n m  (♦) and 50 n m  (■) T4 DNA ligase with 100 n m  substrate 1.  Reactions were run in the standard assay buffer. Each time point represents the average of three experiments, and the  error bars  represent one S.D. The  dashed lines  represent fits by simulation using the chemical rates determined from single turnover reaction of substrate  1 , literature values for Step 1 rates, and diffusion-limited binding of DNA and allowing the rate of product release ( k off ) and the amplitude ( a ) to vary. The best fit was obtained with  a  = 0.51 and  k off  = 0.58 s −1 .
    Figure Legend Snippet: Pre-steady state reactions of 30 n m (♦) and 50 n m (■) T4 DNA ligase with 100 n m substrate 1. Reactions were run in the standard assay buffer. Each time point represents the average of three experiments, and the error bars represent one S.D. The dashed lines represent fits by simulation using the chemical rates determined from single turnover reaction of substrate 1 , literature values for Step 1 rates, and diffusion-limited binding of DNA and allowing the rate of product release ( k off ) and the amplitude ( a ) to vary. The best fit was obtained with a = 0.51 and k off = 0.58 s −1 .

    Techniques Used: Diffusion-based Assay, Binding Assay

    Determination of  k cat  and  k cat / K m  for T4 DNA ligase and nicked substrates.  Shown is reaction of 1 n m  T4 DNA ligase with 1 n m  (○), 2 n m  (*), 5 n m  (×), 10 n m  (△), 20 n m  (♢), and 50 n m  (□) substrate  1  in standard assay buffer at 16 °C ( A ) and 1 n m  T4 DNA ligase (
    Figure Legend Snippet: Determination of k cat and k cat / K m for T4 DNA ligase and nicked substrates. Shown is reaction of 1 n m T4 DNA ligase with 1 n m (○), 2 n m (*), 5 n m (×), 10 n m (△), 20 n m (♢), and 50 n m (□) substrate 1 in standard assay buffer at 16 °C ( A ) and 1 n m T4 DNA ligase (

    Techniques Used:

    6) Product Images from "The yeast high mobility group protein HMO2, a subunit of the chromatin-remodeling complex INO80, binds DNA ends"

    Article Title: The yeast high mobility group protein HMO2, a subunit of the chromatin-remodeling complex INO80, binds DNA ends

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp695

    Interaction of HMO2 with plasmid DNA. ( A ,  B ) Agarose gel retardation of 100 ng plasmid DNA titrated with HMO2. (A) Reactions with supercoiled pGEM5. Lane 1, DNA only, lanes 2–7 with 1.0–6.0 μM HMO2. (B) Reactions with linearized pGEM5. Lane 1, DNA only, lanes 2–6 with 1.0–5.0 μM HMO2. ( C ) HMO2 supercoils relaxed DNA. Lane 1, 100 ng supercoiled pUC18 DNA. Lane 2, nicked pUC18. Lane 3, nicked pUC18 and T4 DNA ligase. Lanes 4–8, nicked DNA and T4 DNA ligase with 100, 500, 1000, 2000 and 3000 nM HMO2.
    Figure Legend Snippet: Interaction of HMO2 with plasmid DNA. ( A , B ) Agarose gel retardation of 100 ng plasmid DNA titrated with HMO2. (A) Reactions with supercoiled pGEM5. Lane 1, DNA only, lanes 2–7 with 1.0–6.0 μM HMO2. (B) Reactions with linearized pGEM5. Lane 1, DNA only, lanes 2–6 with 1.0–5.0 μM HMO2. ( C ) HMO2 supercoils relaxed DNA. Lane 1, 100 ng supercoiled pUC18 DNA. Lane 2, nicked pUC18. Lane 3, nicked pUC18 and T4 DNA ligase. Lanes 4–8, nicked DNA and T4 DNA ligase with 100, 500, 1000, 2000 and 3000 nM HMO2.

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis

    HMO2 prevents ligation of DNA by T4 DNA ligase. ( A ) DNA with overhangs (5′-TA extensions). ( B ) DNA with blunt ends. Lanes 1, 100 ng of DNA (∼4 nM, corresponding to ∼8 nM DNA ends). Lane 2, DNA and T4 DNA ligase. Lanes 3–8, DNA, T4 DNA ligase with 100, 500, 1000, 2000, 3000 and 4000 nM HMO2. Lane 9, DNA, T4 DNA ligase, 4000 nM HMO2 and exonuclease III.
    Figure Legend Snippet: HMO2 prevents ligation of DNA by T4 DNA ligase. ( A ) DNA with overhangs (5′-TA extensions). ( B ) DNA with blunt ends. Lanes 1, 100 ng of DNA (∼4 nM, corresponding to ∼8 nM DNA ends). Lane 2, DNA and T4 DNA ligase. Lanes 3–8, DNA, T4 DNA ligase with 100, 500, 1000, 2000, 3000 and 4000 nM HMO2. Lane 9, DNA, T4 DNA ligase, 4000 nM HMO2 and exonuclease III.

    Techniques Used: Ligation

    HMO1 promotes DNA end-joining, but does not protect DNA from exonucleolytic cleavage. ( A ) HMO1 can promote end-joining of pGEM5 DNA with 2-nt 5′ overhang in presence of T4 DNA ligase. Lane 1, 100 ng DNA only. Lane 2, DNA and T4 DNA ligase. Lanes 3–5, DNA, T4 DNA ligase, and 500, 1000 and 2000 nM HMO1, respectively. ( B ) HMO1 is unable to protect DNA with 2-nt 5′ overhangs from exonuclease III. Lane 1, 100 ng DNA only. Lane 2, DNA and exonuclease III. Lane 3, DNA and 500 nM HMO1. Lanes 4–6, DNA, exonuclease III, and 500, 1000 and 2000 nM HMO1, respectively.
    Figure Legend Snippet: HMO1 promotes DNA end-joining, but does not protect DNA from exonucleolytic cleavage. ( A ) HMO1 can promote end-joining of pGEM5 DNA with 2-nt 5′ overhang in presence of T4 DNA ligase. Lane 1, 100 ng DNA only. Lane 2, DNA and T4 DNA ligase. Lanes 3–5, DNA, T4 DNA ligase, and 500, 1000 and 2000 nM HMO1, respectively. ( B ) HMO1 is unable to protect DNA with 2-nt 5′ overhangs from exonuclease III. Lane 1, 100 ng DNA only. Lane 2, DNA and exonuclease III. Lane 3, DNA and 500 nM HMO1. Lanes 4–6, DNA, exonuclease III, and 500, 1000 and 2000 nM HMO1, respectively.

    Techniques Used:

    DNA protection by HMO2 depends on DNA length and sequence of DNA overhangs. ( A ) DNA with G+C-containing overhangs is not protected by HMO2. Lanes 1–4, DNA with 5′-CATG extensions (∼2 nM), lanes 5–8, DNA with 5′-TA extensions (∼4 nM). Lanes 1 and 5, DNA only. Lanes 2 and 6, DNA treated with exonuclease III for 1 h. Lanes 3 and 7, DNA and 2000 nM HMO2. Lanes 4 and 8, DNA with 2000 nM HMO2 incubated with exonuclease III for 1 h. Note in lane 8 the appearance of a product with lower mobility. Only the two largest fragments of BspHI-digested pET5a are shown in lanes 1–4. ( B ) Ligation of DNA with 5′-CATG extension (∼2 nM). Lane 1, DNA only. Lane 2, DNA and T4 DNA ligase. Lane 3, DNA, T4 DNA ligase and 2.5 µM HMO2. ( C ) Length dependence of DNA protection by HMO2. Lane 1, DNA with 4-nt 5′ overhangs. Lane 2, DNA treated with exonuclease III for 1 h. Lane 3, DNA and 2000 nM HMO2. Lane 4, DNA incubated with HMO2 and exonuclease III for 1 h. ( D ) HMO2 can end-join 105 bp DNA in presence of T4 DNA ligase. Lane 1, 100 fmol of 105 bp DNA. Lane 2, 105 bp DNA and T4 DNA ligase. Lanes 3–5, 105 bp DNA, T4 DNA ligase and 100, 250 and 500 nM HMO2. Lane 6, 105 bp DNA, T4 DNA ligase and 100 nM  B. subtilis  HU (HBsu). Lane 7, 105 bp DNA, T4 DNA ligase, 100 nM  B. subtilis  HU and exonuclease III. Lane 8, 105 bp DNA, T4 DNA ligase, 250 nM HMO2 and exonuclease III.
    Figure Legend Snippet: DNA protection by HMO2 depends on DNA length and sequence of DNA overhangs. ( A ) DNA with G+C-containing overhangs is not protected by HMO2. Lanes 1–4, DNA with 5′-CATG extensions (∼2 nM), lanes 5–8, DNA with 5′-TA extensions (∼4 nM). Lanes 1 and 5, DNA only. Lanes 2 and 6, DNA treated with exonuclease III for 1 h. Lanes 3 and 7, DNA and 2000 nM HMO2. Lanes 4 and 8, DNA with 2000 nM HMO2 incubated with exonuclease III for 1 h. Note in lane 8 the appearance of a product with lower mobility. Only the two largest fragments of BspHI-digested pET5a are shown in lanes 1–4. ( B ) Ligation of DNA with 5′-CATG extension (∼2 nM). Lane 1, DNA only. Lane 2, DNA and T4 DNA ligase. Lane 3, DNA, T4 DNA ligase and 2.5 µM HMO2. ( C ) Length dependence of DNA protection by HMO2. Lane 1, DNA with 4-nt 5′ overhangs. Lane 2, DNA treated with exonuclease III for 1 h. Lane 3, DNA and 2000 nM HMO2. Lane 4, DNA incubated with HMO2 and exonuclease III for 1 h. ( D ) HMO2 can end-join 105 bp DNA in presence of T4 DNA ligase. Lane 1, 100 fmol of 105 bp DNA. Lane 2, 105 bp DNA and T4 DNA ligase. Lanes 3–5, 105 bp DNA, T4 DNA ligase and 100, 250 and 500 nM HMO2. Lane 6, 105 bp DNA, T4 DNA ligase and 100 nM B. subtilis HU (HBsu). Lane 7, 105 bp DNA, T4 DNA ligase, 100 nM B. subtilis HU and exonuclease III. Lane 8, 105 bp DNA, T4 DNA ligase, 250 nM HMO2 and exonuclease III.

    Techniques Used: Sequencing, Incubation, Ligation

    7) Product Images from "The yeast high mobility group protein HMO2, a subunit of the chromatin-remodeling complex INO80, binds DNA ends"

    Article Title: The yeast high mobility group protein HMO2, a subunit of the chromatin-remodeling complex INO80, binds DNA ends

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp695

    Interaction of HMO2 with plasmid DNA. ( A ,  B ) Agarose gel retardation of 100 ng plasmid DNA titrated with HMO2. (A) Reactions with supercoiled pGEM5. Lane 1, DNA only, lanes 2–7 with 1.0–6.0 μM HMO2. (B) Reactions with linearized pGEM5. Lane 1, DNA only, lanes 2–6 with 1.0–5.0 μM HMO2. ( C ) HMO2 supercoils relaxed DNA. Lane 1, 100 ng supercoiled pUC18 DNA. Lane 2, nicked pUC18. Lane 3, nicked pUC18 and T4 DNA ligase. Lanes 4–8, nicked DNA and T4 DNA ligase with 100, 500, 1000, 2000 and 3000 nM HMO2.
    Figure Legend Snippet: Interaction of HMO2 with plasmid DNA. ( A , B ) Agarose gel retardation of 100 ng plasmid DNA titrated with HMO2. (A) Reactions with supercoiled pGEM5. Lane 1, DNA only, lanes 2–7 with 1.0–6.0 μM HMO2. (B) Reactions with linearized pGEM5. Lane 1, DNA only, lanes 2–6 with 1.0–5.0 μM HMO2. ( C ) HMO2 supercoils relaxed DNA. Lane 1, 100 ng supercoiled pUC18 DNA. Lane 2, nicked pUC18. Lane 3, nicked pUC18 and T4 DNA ligase. Lanes 4–8, nicked DNA and T4 DNA ligase with 100, 500, 1000, 2000 and 3000 nM HMO2.

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis

    HMO2 prevents ligation of DNA by T4 DNA ligase. ( A ) DNA with overhangs (5′-TA extensions). ( B ) DNA with blunt ends. Lanes 1, 100 ng of DNA (∼4 nM, corresponding to ∼8 nM DNA ends). Lane 2, DNA and T4 DNA ligase. Lanes 3–8, DNA, T4 DNA ligase with 100, 500, 1000, 2000, 3000 and 4000 nM HMO2. Lane 9, DNA, T4 DNA ligase, 4000 nM HMO2 and exonuclease III.
    Figure Legend Snippet: HMO2 prevents ligation of DNA by T4 DNA ligase. ( A ) DNA with overhangs (5′-TA extensions). ( B ) DNA with blunt ends. Lanes 1, 100 ng of DNA (∼4 nM, corresponding to ∼8 nM DNA ends). Lane 2, DNA and T4 DNA ligase. Lanes 3–8, DNA, T4 DNA ligase with 100, 500, 1000, 2000, 3000 and 4000 nM HMO2. Lane 9, DNA, T4 DNA ligase, 4000 nM HMO2 and exonuclease III.

    Techniques Used: Ligation

    HMO1 promotes DNA end-joining, but does not protect DNA from exonucleolytic cleavage. ( A ) HMO1 can promote end-joining of pGEM5 DNA with 2-nt 5′ overhang in presence of T4 DNA ligase. Lane 1, 100 ng DNA only. Lane 2, DNA and T4 DNA ligase. Lanes 3–5, DNA, T4 DNA ligase, and 500, 1000 and 2000 nM HMO1, respectively. ( B ) HMO1 is unable to protect DNA with 2-nt 5′ overhangs from exonuclease III. Lane 1, 100 ng DNA only. Lane 2, DNA and exonuclease III. Lane 3, DNA and 500 nM HMO1. Lanes 4–6, DNA, exonuclease III, and 500, 1000 and 2000 nM HMO1, respectively.
    Figure Legend Snippet: HMO1 promotes DNA end-joining, but does not protect DNA from exonucleolytic cleavage. ( A ) HMO1 can promote end-joining of pGEM5 DNA with 2-nt 5′ overhang in presence of T4 DNA ligase. Lane 1, 100 ng DNA only. Lane 2, DNA and T4 DNA ligase. Lanes 3–5, DNA, T4 DNA ligase, and 500, 1000 and 2000 nM HMO1, respectively. ( B ) HMO1 is unable to protect DNA with 2-nt 5′ overhangs from exonuclease III. Lane 1, 100 ng DNA only. Lane 2, DNA and exonuclease III. Lane 3, DNA and 500 nM HMO1. Lanes 4–6, DNA, exonuclease III, and 500, 1000 and 2000 nM HMO1, respectively.

    Techniques Used:

    DNA protection by HMO2 depends on DNA length and sequence of DNA overhangs. ( A ) DNA with G+C-containing overhangs is not protected by HMO2. Lanes 1–4, DNA with 5′-CATG extensions (∼2 nM), lanes 5–8, DNA with 5′-TA extensions (∼4 nM). Lanes 1 and 5, DNA only. Lanes 2 and 6, DNA treated with exonuclease III for 1 h. Lanes 3 and 7, DNA and 2000 nM HMO2. Lanes 4 and 8, DNA with 2000 nM HMO2 incubated with exonuclease III for 1 h. Note in lane 8 the appearance of a product with lower mobility. Only the two largest fragments of BspHI-digested pET5a are shown in lanes 1–4. ( B ) Ligation of DNA with 5′-CATG extension (∼2 nM). Lane 1, DNA only. Lane 2, DNA and T4 DNA ligase. Lane 3, DNA, T4 DNA ligase and 2.5 µM HMO2. ( C ) Length dependence of DNA protection by HMO2. Lane 1, DNA with 4-nt 5′ overhangs. Lane 2, DNA treated with exonuclease III for 1 h. Lane 3, DNA and 2000 nM HMO2. Lane 4, DNA incubated with HMO2 and exonuclease III for 1 h. ( D ) HMO2 can end-join 105 bp DNA in presence of T4 DNA ligase. Lane 1, 100 fmol of 105 bp DNA. Lane 2, 105 bp DNA and T4 DNA ligase. Lanes 3–5, 105 bp DNA, T4 DNA ligase and 100, 250 and 500 nM HMO2. Lane 6, 105 bp DNA, T4 DNA ligase and 100 nM  B. subtilis  HU (HBsu). Lane 7, 105 bp DNA, T4 DNA ligase, 100 nM  B. subtilis  HU and exonuclease III. Lane 8, 105 bp DNA, T4 DNA ligase, 250 nM HMO2 and exonuclease III.
    Figure Legend Snippet: DNA protection by HMO2 depends on DNA length and sequence of DNA overhangs. ( A ) DNA with G+C-containing overhangs is not protected by HMO2. Lanes 1–4, DNA with 5′-CATG extensions (∼2 nM), lanes 5–8, DNA with 5′-TA extensions (∼4 nM). Lanes 1 and 5, DNA only. Lanes 2 and 6, DNA treated with exonuclease III for 1 h. Lanes 3 and 7, DNA and 2000 nM HMO2. Lanes 4 and 8, DNA with 2000 nM HMO2 incubated with exonuclease III for 1 h. Note in lane 8 the appearance of a product with lower mobility. Only the two largest fragments of BspHI-digested pET5a are shown in lanes 1–4. ( B ) Ligation of DNA with 5′-CATG extension (∼2 nM). Lane 1, DNA only. Lane 2, DNA and T4 DNA ligase. Lane 3, DNA, T4 DNA ligase and 2.5 µM HMO2. ( C ) Length dependence of DNA protection by HMO2. Lane 1, DNA with 4-nt 5′ overhangs. Lane 2, DNA treated with exonuclease III for 1 h. Lane 3, DNA and 2000 nM HMO2. Lane 4, DNA incubated with HMO2 and exonuclease III for 1 h. ( D ) HMO2 can end-join 105 bp DNA in presence of T4 DNA ligase. Lane 1, 100 fmol of 105 bp DNA. Lane 2, 105 bp DNA and T4 DNA ligase. Lanes 3–5, 105 bp DNA, T4 DNA ligase and 100, 250 and 500 nM HMO2. Lane 6, 105 bp DNA, T4 DNA ligase and 100 nM B. subtilis HU (HBsu). Lane 7, 105 bp DNA, T4 DNA ligase, 100 nM B. subtilis HU and exonuclease III. Lane 8, 105 bp DNA, T4 DNA ligase, 250 nM HMO2 and exonuclease III.

    Techniques Used: Sequencing, Incubation, Ligation

    8) Product Images from "Structure-seq2: sensitive and accurate genome-wide profiling of RNA structure in vivo"

    Article Title: Structure-seq2: sensitive and accurate genome-wide profiling of RNA structure in vivo

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx533

    Structure-seq2 leads to a lower ligation bias. ( A ) After RT (Figure   1 , step 1A/1B), excess of the 27 nt primer (blue, top, right) is still present in the solution. During ligation (Figure   1 , step 3A/3B), this primer can also ligate to the 40 nt hairpin adaptor (pink) to form an unwanted 67 nt by-product which has no insert and so results in sequencing reads with no utility. ( B ) The complement of the first nucleotide after the adaptor sequence read during sequencing is the nucleotide that ligated to the adaptor. Our new T4 DNA ligase-based method (green, –DMS and pink, +DMS) substantially decreases ligation bias as compared to the previous Circligase-based method (blue). Percentages equaling the transcriptomic distribution of the four nucleotides (black) are ideal.
    Figure Legend Snippet: Structure-seq2 leads to a lower ligation bias. ( A ) After RT (Figure 1 , step 1A/1B), excess of the 27 nt primer (blue, top, right) is still present in the solution. During ligation (Figure 1 , step 3A/3B), this primer can also ligate to the 40 nt hairpin adaptor (pink) to form an unwanted 67 nt by-product which has no insert and so results in sequencing reads with no utility. ( B ) The complement of the first nucleotide after the adaptor sequence read during sequencing is the nucleotide that ligated to the adaptor. Our new T4 DNA ligase-based method (green, –DMS and pink, +DMS) substantially decreases ligation bias as compared to the previous Circligase-based method (blue). Percentages equaling the transcriptomic distribution of the four nucleotides (black) are ideal.

    Techniques Used: Ligation, Sequencing

    Two versions of Structure-seq2 produce high quality data. In Structure-seq2, RNA (kelly green) is first modified by DMS or another chemical that can be read-out through reverse transcription. The RNA is then prepared for Illumina NGS sequencing by conversion to cDNA (Step 1A/1B, blue), ligating an adaptor (Step 3A/3B), and amplifying the products while incorporating TruSeq primer sequences (Step 5A/5B). In order to increase library quality, numerous improvements were made to the original Structure-seq protocol (boxed). These include performing the ligation with a hairpin adaptor and T4 DNA ligase (Step 3A/3B; pink) (  10 ), and adding various purification steps to remove a deleterious by-product (Figure   2A ). We present two options for purification: PAGE purification ( A ) or a biotin–streptavidin pull down ( B ). In the PAGE purification method, an additional PAGE purification step is added after reverse transcription (Step 2A). In the biotin–streptavidin pull down method, biotinylated dNTPs (cyan) are incorporated into the extended product during reverse transcription (Step 1B) and are purified via a magnetic streptavidin pull down after reverse transcription (Step 2B) and after ligation (Step 4B). There is also a common, final PAGE purification step following amplification (Step 5A/5B). Finally, a custom sequencing primer (light green) is used during sequencing (Step 7A/7B) to further provide high quality data.   Supplementary Figure S1  is a version of this figure with all the nucleotides shown explicitly.
    Figure Legend Snippet: Two versions of Structure-seq2 produce high quality data. In Structure-seq2, RNA (kelly green) is first modified by DMS or another chemical that can be read-out through reverse transcription. The RNA is then prepared for Illumina NGS sequencing by conversion to cDNA (Step 1A/1B, blue), ligating an adaptor (Step 3A/3B), and amplifying the products while incorporating TruSeq primer sequences (Step 5A/5B). In order to increase library quality, numerous improvements were made to the original Structure-seq protocol (boxed). These include performing the ligation with a hairpin adaptor and T4 DNA ligase (Step 3A/3B; pink) ( 10 ), and adding various purification steps to remove a deleterious by-product (Figure 2A ). We present two options for purification: PAGE purification ( A ) or a biotin–streptavidin pull down ( B ). In the PAGE purification method, an additional PAGE purification step is added after reverse transcription (Step 2A). In the biotin–streptavidin pull down method, biotinylated dNTPs (cyan) are incorporated into the extended product during reverse transcription (Step 1B) and are purified via a magnetic streptavidin pull down after reverse transcription (Step 2B) and after ligation (Step 4B). There is also a common, final PAGE purification step following amplification (Step 5A/5B). Finally, a custom sequencing primer (light green) is used during sequencing (Step 7A/7B) to further provide high quality data. Supplementary Figure S1 is a version of this figure with all the nucleotides shown explicitly.

    Techniques Used: Modification, Next-Generation Sequencing, Sequencing, Ligation, Purification, Polyacrylamide Gel Electrophoresis, Amplification

    9) Product Images from ""

    Article Title:

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.284992

    Reaction of T4 DNA ligase with substrate 1 ( A ) and adenylylated substrate 1A ( B ) under single turnover conditions.  Each reaction was run with 500 n m  ligase and 100 n m  substrate in the standard ATP-free assay buffer. Ligase that was  > 95% adenylylated was used for  A , and
    Figure Legend Snippet: Reaction of T4 DNA ligase with substrate 1 ( A ) and adenylylated substrate 1A ( B ) under single turnover conditions. Each reaction was run with 500 n m ligase and 100 n m substrate in the standard ATP-free assay buffer. Ligase that was > 95% adenylylated was used for A , and

    Techniques Used:

    Pre-steady state reactions of 30 n m  (♦) and 50 n m  (■) T4 DNA ligase with 100 n m  substrate 1.  Reactions were run in the standard assay buffer. Each time point represents the average of three experiments, and the  error bars  represent one S.D. The  dashed lines  represent fits by simulation using the chemical rates determined from single turnover reaction of substrate  1 , literature values for Step 1 rates, and diffusion-limited binding of DNA and allowing the rate of product release ( k off ) and the amplitude ( a ) to vary. The best fit was obtained with  a  = 0.51 and  k off  = 0.58 s −1 .
    Figure Legend Snippet: Pre-steady state reactions of 30 n m (♦) and 50 n m (■) T4 DNA ligase with 100 n m substrate 1. Reactions were run in the standard assay buffer. Each time point represents the average of three experiments, and the error bars represent one S.D. The dashed lines represent fits by simulation using the chemical rates determined from single turnover reaction of substrate 1 , literature values for Step 1 rates, and diffusion-limited binding of DNA and allowing the rate of product release ( k off ) and the amplitude ( a ) to vary. The best fit was obtained with a = 0.51 and k off = 0.58 s −1 .

    Techniques Used: Diffusion-based Assay, Binding Assay

    Determination of  k cat  and  k cat / K m  for T4 DNA ligase and nicked substrates.  Shown is reaction of 1 n m  T4 DNA ligase with 1 n m  (○), 2 n m  (*), 5 n m  (×), 10 n m  (△), 20 n m  (♢), and 50 n m  (□) substrate  1  in standard assay buffer at 16 °C ( A ) and 1 n m  T4 DNA ligase (
    Figure Legend Snippet: Determination of k cat and k cat / K m for T4 DNA ligase and nicked substrates. Shown is reaction of 1 n m T4 DNA ligase with 1 n m (○), 2 n m (*), 5 n m (×), 10 n m (△), 20 n m (♢), and 50 n m (□) substrate 1 in standard assay buffer at 16 °C ( A ) and 1 n m T4 DNA ligase (

    Techniques Used:

    10) Product Images from ""

    Article Title:

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.284992

    Reaction of T4 DNA ligase with substrate 1 ( A ) and adenylylated substrate 1A ( B ) under single turnover conditions.  Each reaction was run with 500 n m  ligase and 100 n m  substrate in the standard ATP-free assay buffer. Ligase that was  > 95% adenylylated was used for  A , and
    Figure Legend Snippet: Reaction of T4 DNA ligase with substrate 1 ( A ) and adenylylated substrate 1A ( B ) under single turnover conditions. Each reaction was run with 500 n m ligase and 100 n m substrate in the standard ATP-free assay buffer. Ligase that was > 95% adenylylated was used for A , and

    Techniques Used:

    Pre-steady state reactions of 30 n m  (♦) and 50 n m  (■) T4 DNA ligase with 100 n m  substrate 1.  Reactions were run in the standard assay buffer. Each time point represents the average of three experiments, and the  error bars  represent one S.D. The  dashed lines  represent fits by simulation using the chemical rates determined from single turnover reaction of substrate  1 , literature values for Step 1 rates, and diffusion-limited binding of DNA and allowing the rate of product release ( k off ) and the amplitude ( a ) to vary. The best fit was obtained with  a  = 0.51 and  k off  = 0.58 s −1 .
    Figure Legend Snippet: Pre-steady state reactions of 30 n m (♦) and 50 n m (■) T4 DNA ligase with 100 n m substrate 1. Reactions were run in the standard assay buffer. Each time point represents the average of three experiments, and the error bars represent one S.D. The dashed lines represent fits by simulation using the chemical rates determined from single turnover reaction of substrate 1 , literature values for Step 1 rates, and diffusion-limited binding of DNA and allowing the rate of product release ( k off ) and the amplitude ( a ) to vary. The best fit was obtained with a = 0.51 and k off = 0.58 s −1 .

    Techniques Used: Diffusion-based Assay, Binding Assay

    Determination of  k cat  and  k cat / K m  for T4 DNA ligase and nicked substrates.  Shown is reaction of 1 n m  T4 DNA ligase with 1 n m  (○), 2 n m  (*), 5 n m  (×), 10 n m  (△), 20 n m  (♢), and 50 n m  (□) substrate  1  in standard assay buffer at 16 °C ( A ) and 1 n m  T4 DNA ligase (
    Figure Legend Snippet: Determination of k cat and k cat / K m for T4 DNA ligase and nicked substrates. Shown is reaction of 1 n m T4 DNA ligase with 1 n m (○), 2 n m (*), 5 n m (×), 10 n m (△), 20 n m (♢), and 50 n m (□) substrate 1 in standard assay buffer at 16 °C ( A ) and 1 n m T4 DNA ligase (

    Techniques Used:

    11) Product Images from "Probing transient protein-mediated DNA linkages using nanoconfinement"

    Article Title: Probing transient protein-mediated DNA linkages using nanoconfinement

    Journal:

    doi: 10.1063/1.4882775

    AFM images of DNA-DNA crossings. (a) Bare DNA (3.8 kbp). (b) and (c) DNA with T4 DNA ligase andATP. Solid arrows indicate higher crossings consistent with ligase binding, outlined arrows indicateshallower crossings consistent with bare DNA.
    Figure Legend Snippet: AFM images of DNA-DNA crossings. (a) Bare DNA (3.8 kbp). (b) and (c) DNA with T4 DNA ligase andATP. Solid arrows indicate higher crossings consistent with ligase binding, outlined arrows indicateshallower crossings consistent with bare DNA.

    Techniques Used: Binding Assay

    Mean aligned DNA molecule loop lengths as function of time for 22 molecules per dataset withtheir linear fits. Bare λ-DNA (blue), λ-DNA with T4 DNA ligase (green), and λ-DNA with T4 DNA ligaseand ATP (red).
    Figure Legend Snippet: Mean aligned DNA molecule loop lengths as function of time for 22 molecules per dataset withtheir linear fits. Bare λ-DNA (blue), λ-DNA with T4 DNA ligase (green), and λ-DNA with T4 DNA ligaseand ATP (red).

    Techniques Used:

    Histogram of end-to-end lengths of extended DNA molecules, bare λ-DNA (solid bars), λ-DNA with T4DNA ligase (gray bars), and λ-DNA with T4 DNA ligase and ATP (white bars). A Gaussian was fit toeach distribution to determine the
    Figure Legend Snippet: Histogram of end-to-end lengths of extended DNA molecules, bare λ-DNA (solid bars), λ-DNA with T4DNA ligase (gray bars), and λ-DNA with T4 DNA ligase and ATP (white bars). A Gaussian was fit toeach distribution to determine the

    Techniques Used:

    Histograms of heights of DNA-DNA crossings. (a) Bare DNA (N = 41). (b) DNA with T4 DNA ligase andATP (N = 174). The red dotted line corresponds to unoccupied crossings, the blue dashed line tooccupied crossings, and the
    Figure Legend Snippet: Histograms of heights of DNA-DNA crossings. (a) Bare DNA (N = 41). (b) DNA with T4 DNA ligase andATP (N = 174). The red dotted line corresponds to unoccupied crossings, the blue dashed line tooccupied crossings, and the

    Techniques Used:

    12) Product Images from ""

    Article Title:

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.284992

    Reaction of T4 DNA ligase with substrate 1 ( A ) and adenylylated substrate 1A ( B ) under single turnover conditions.  Each reaction was run with 500 n m  ligase and 100 n m  substrate in the standard ATP-free assay buffer. Ligase that was  > 95% adenylylated was used for  A , and
    Figure Legend Snippet: Reaction of T4 DNA ligase with substrate 1 ( A ) and adenylylated substrate 1A ( B ) under single turnover conditions. Each reaction was run with 500 n m ligase and 100 n m substrate in the standard ATP-free assay buffer. Ligase that was > 95% adenylylated was used for A , and

    Techniques Used:

    Pre-steady state reactions of 30 n m  (♦) and 50 n m  (■) T4 DNA ligase with 100 n m  substrate 1.  Reactions were run in the standard assay buffer. Each time point represents the average of three experiments, and the  error bars  represent one S.D. The  dashed lines  represent fits by simulation using the chemical rates determined from single turnover reaction of substrate  1 , literature values for Step 1 rates, and diffusion-limited binding of DNA and allowing the rate of product release ( k off ) and the amplitude ( a ) to vary. The best fit was obtained with  a  = 0.51 and  k off  = 0.58 s −1 .
    Figure Legend Snippet: Pre-steady state reactions of 30 n m (♦) and 50 n m (■) T4 DNA ligase with 100 n m substrate 1. Reactions were run in the standard assay buffer. Each time point represents the average of three experiments, and the error bars represent one S.D. The dashed lines represent fits by simulation using the chemical rates determined from single turnover reaction of substrate 1 , literature values for Step 1 rates, and diffusion-limited binding of DNA and allowing the rate of product release ( k off ) and the amplitude ( a ) to vary. The best fit was obtained with a = 0.51 and k off = 0.58 s −1 .

    Techniques Used: Diffusion-based Assay, Binding Assay

    Determination of  k cat  and  k cat / K m  for T4 DNA ligase and nicked substrates.  Shown is reaction of 1 n m  T4 DNA ligase with 1 n m  (○), 2 n m  (*), 5 n m  (×), 10 n m  (△), 20 n m  (♢), and 50 n m  (□) substrate  1  in standard assay buffer at 16 °C ( A ) and 1 n m  T4 DNA ligase (
    Figure Legend Snippet: Determination of k cat and k cat / K m for T4 DNA ligase and nicked substrates. Shown is reaction of 1 n m T4 DNA ligase with 1 n m (○), 2 n m (*), 5 n m (×), 10 n m (△), 20 n m (♢), and 50 n m (□) substrate 1 in standard assay buffer at 16 °C ( A ) and 1 n m T4 DNA ligase (

    Techniques Used:

    13) Product Images from ""

    Article Title:

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.284992

    Reaction of T4 DNA ligase with substrate 1 ( A ) and adenylylated substrate 1A ( B ) under single turnover conditions.  Each reaction was run with 500 n m  ligase and 100 n m  substrate in the standard ATP-free assay buffer. Ligase that was  > 95% adenylylated was used for  A , and
    Figure Legend Snippet: Reaction of T4 DNA ligase with substrate 1 ( A ) and adenylylated substrate 1A ( B ) under single turnover conditions. Each reaction was run with 500 n m ligase and 100 n m substrate in the standard ATP-free assay buffer. Ligase that was > 95% adenylylated was used for A , and

    Techniques Used:

    Pre-steady state reactions of 30 n m  (♦) and 50 n m  (■) T4 DNA ligase with 100 n m  substrate 1.  Reactions were run in the standard assay buffer. Each time point represents the average of three experiments, and the  error bars  represent one S.D. The  dashed lines  represent fits by simulation using the chemical rates determined from single turnover reaction of substrate  1 , literature values for Step 1 rates, and diffusion-limited binding of DNA and allowing the rate of product release ( k off ) and the amplitude ( a ) to vary. The best fit was obtained with  a  = 0.51 and  k off  = 0.58 s −1 .
    Figure Legend Snippet: Pre-steady state reactions of 30 n m (♦) and 50 n m (■) T4 DNA ligase with 100 n m substrate 1. Reactions were run in the standard assay buffer. Each time point represents the average of three experiments, and the error bars represent one S.D. The dashed lines represent fits by simulation using the chemical rates determined from single turnover reaction of substrate 1 , literature values for Step 1 rates, and diffusion-limited binding of DNA and allowing the rate of product release ( k off ) and the amplitude ( a ) to vary. The best fit was obtained with a = 0.51 and k off = 0.58 s −1 .

    Techniques Used: Diffusion-based Assay, Binding Assay

    Determination of  k cat  and  k cat / K m  for T4 DNA ligase and nicked substrates.  Shown is reaction of 1 n m  T4 DNA ligase with 1 n m  (○), 2 n m  (*), 5 n m  (×), 10 n m  (△), 20 n m  (♢), and 50 n m  (□) substrate  1  in standard assay buffer at 16 °C ( A ) and 1 n m  T4 DNA ligase (
    Figure Legend Snippet: Determination of k cat and k cat / K m for T4 DNA ligase and nicked substrates. Shown is reaction of 1 n m T4 DNA ligase with 1 n m (○), 2 n m (*), 5 n m (×), 10 n m (△), 20 n m (♢), and 50 n m (□) substrate 1 in standard assay buffer at 16 °C ( A ) and 1 n m T4 DNA ligase (

    Techniques Used:

    14) Product Images from "The yeast high mobility group protein HMO2, a subunit of the chromatin-remodeling complex INO80, binds DNA ends"

    Article Title: The yeast high mobility group protein HMO2, a subunit of the chromatin-remodeling complex INO80, binds DNA ends

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp695

    Interaction of HMO2 with plasmid DNA. ( A ,  B ) Agarose gel retardation of 100 ng plasmid DNA titrated with HMO2. (A) Reactions with supercoiled pGEM5. Lane 1, DNA only, lanes 2–7 with 1.0–6.0 μM HMO2. (B) Reactions with linearized pGEM5. Lane 1, DNA only, lanes 2–6 with 1.0–5.0 μM HMO2. ( C ) HMO2 supercoils relaxed DNA. Lane 1, 100 ng supercoiled pUC18 DNA. Lane 2, nicked pUC18. Lane 3, nicked pUC18 and T4 DNA ligase. Lanes 4–8, nicked DNA and T4 DNA ligase with 100, 500, 1000, 2000 and 3000 nM HMO2.
    Figure Legend Snippet: Interaction of HMO2 with plasmid DNA. ( A , B ) Agarose gel retardation of 100 ng plasmid DNA titrated with HMO2. (A) Reactions with supercoiled pGEM5. Lane 1, DNA only, lanes 2–7 with 1.0–6.0 μM HMO2. (B) Reactions with linearized pGEM5. Lane 1, DNA only, lanes 2–6 with 1.0–5.0 μM HMO2. ( C ) HMO2 supercoils relaxed DNA. Lane 1, 100 ng supercoiled pUC18 DNA. Lane 2, nicked pUC18. Lane 3, nicked pUC18 and T4 DNA ligase. Lanes 4–8, nicked DNA and T4 DNA ligase with 100, 500, 1000, 2000 and 3000 nM HMO2.

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis

    HMO2 prevents ligation of DNA by T4 DNA ligase. ( A ) DNA with overhangs (5′-TA extensions). ( B ) DNA with blunt ends. Lanes 1, 100 ng of DNA (∼4 nM, corresponding to ∼8 nM DNA ends). Lane 2, DNA and T4 DNA ligase. Lanes 3–8, DNA, T4 DNA ligase with 100, 500, 1000, 2000, 3000 and 4000 nM HMO2. Lane 9, DNA, T4 DNA ligase, 4000 nM HMO2 and exonuclease III.
    Figure Legend Snippet: HMO2 prevents ligation of DNA by T4 DNA ligase. ( A ) DNA with overhangs (5′-TA extensions). ( B ) DNA with blunt ends. Lanes 1, 100 ng of DNA (∼4 nM, corresponding to ∼8 nM DNA ends). Lane 2, DNA and T4 DNA ligase. Lanes 3–8, DNA, T4 DNA ligase with 100, 500, 1000, 2000, 3000 and 4000 nM HMO2. Lane 9, DNA, T4 DNA ligase, 4000 nM HMO2 and exonuclease III.

    Techniques Used: Ligation

    HMO1 promotes DNA end-joining, but does not protect DNA from exonucleolytic cleavage. ( A ) HMO1 can promote end-joining of pGEM5 DNA with 2-nt 5′ overhang in presence of T4 DNA ligase. Lane 1, 100 ng DNA only. Lane 2, DNA and T4 DNA ligase. Lanes 3–5, DNA, T4 DNA ligase, and 500, 1000 and 2000 nM HMO1, respectively. ( B ) HMO1 is unable to protect DNA with 2-nt 5′ overhangs from exonuclease III. Lane 1, 100 ng DNA only. Lane 2, DNA and exonuclease III. Lane 3, DNA and 500 nM HMO1. Lanes 4–6, DNA, exonuclease III, and 500, 1000 and 2000 nM HMO1, respectively.
    Figure Legend Snippet: HMO1 promotes DNA end-joining, but does not protect DNA from exonucleolytic cleavage. ( A ) HMO1 can promote end-joining of pGEM5 DNA with 2-nt 5′ overhang in presence of T4 DNA ligase. Lane 1, 100 ng DNA only. Lane 2, DNA and T4 DNA ligase. Lanes 3–5, DNA, T4 DNA ligase, and 500, 1000 and 2000 nM HMO1, respectively. ( B ) HMO1 is unable to protect DNA with 2-nt 5′ overhangs from exonuclease III. Lane 1, 100 ng DNA only. Lane 2, DNA and exonuclease III. Lane 3, DNA and 500 nM HMO1. Lanes 4–6, DNA, exonuclease III, and 500, 1000 and 2000 nM HMO1, respectively.

    Techniques Used:

    DNA protection by HMO2 depends on DNA length and sequence of DNA overhangs. ( A ) DNA with G+C-containing overhangs is not protected by HMO2. Lanes 1–4, DNA with 5′-CATG extensions (∼2 nM), lanes 5–8, DNA with 5′-TA extensions (∼4 nM). Lanes 1 and 5, DNA only. Lanes 2 and 6, DNA treated with exonuclease III for 1 h. Lanes 3 and 7, DNA and 2000 nM HMO2. Lanes 4 and 8, DNA with 2000 nM HMO2 incubated with exonuclease III for 1 h. Note in lane 8 the appearance of a product with lower mobility. Only the two largest fragments of BspHI-digested pET5a are shown in lanes 1–4. ( B ) Ligation of DNA with 5′-CATG extension (∼2 nM). Lane 1, DNA only. Lane 2, DNA and T4 DNA ligase. Lane 3, DNA, T4 DNA ligase and 2.5 µM HMO2. ( C ) Length dependence of DNA protection by HMO2. Lane 1, DNA with 4-nt 5′ overhangs. Lane 2, DNA treated with exonuclease III for 1 h. Lane 3, DNA and 2000 nM HMO2. Lane 4, DNA incubated with HMO2 and exonuclease III for 1 h. ( D ) HMO2 can end-join 105 bp DNA in presence of T4 DNA ligase. Lane 1, 100 fmol of 105 bp DNA. Lane 2, 105 bp DNA and T4 DNA ligase. Lanes 3–5, 105 bp DNA, T4 DNA ligase and 100, 250 and 500 nM HMO2. Lane 6, 105 bp DNA, T4 DNA ligase and 100 nM  B. subtilis  HU (HBsu). Lane 7, 105 bp DNA, T4 DNA ligase, 100 nM  B. subtilis  HU and exonuclease III. Lane 8, 105 bp DNA, T4 DNA ligase, 250 nM HMO2 and exonuclease III.
    Figure Legend Snippet: DNA protection by HMO2 depends on DNA length and sequence of DNA overhangs. ( A ) DNA with G+C-containing overhangs is not protected by HMO2. Lanes 1–4, DNA with 5′-CATG extensions (∼2 nM), lanes 5–8, DNA with 5′-TA extensions (∼4 nM). Lanes 1 and 5, DNA only. Lanes 2 and 6, DNA treated with exonuclease III for 1 h. Lanes 3 and 7, DNA and 2000 nM HMO2. Lanes 4 and 8, DNA with 2000 nM HMO2 incubated with exonuclease III for 1 h. Note in lane 8 the appearance of a product with lower mobility. Only the two largest fragments of BspHI-digested pET5a are shown in lanes 1–4. ( B ) Ligation of DNA with 5′-CATG extension (∼2 nM). Lane 1, DNA only. Lane 2, DNA and T4 DNA ligase. Lane 3, DNA, T4 DNA ligase and 2.5 µM HMO2. ( C ) Length dependence of DNA protection by HMO2. Lane 1, DNA with 4-nt 5′ overhangs. Lane 2, DNA treated with exonuclease III for 1 h. Lane 3, DNA and 2000 nM HMO2. Lane 4, DNA incubated with HMO2 and exonuclease III for 1 h. ( D ) HMO2 can end-join 105 bp DNA in presence of T4 DNA ligase. Lane 1, 100 fmol of 105 bp DNA. Lane 2, 105 bp DNA and T4 DNA ligase. Lanes 3–5, 105 bp DNA, T4 DNA ligase and 100, 250 and 500 nM HMO2. Lane 6, 105 bp DNA, T4 DNA ligase and 100 nM B. subtilis HU (HBsu). Lane 7, 105 bp DNA, T4 DNA ligase, 100 nM B. subtilis HU and exonuclease III. Lane 8, 105 bp DNA, T4 DNA ligase, 250 nM HMO2 and exonuclease III.

    Techniques Used: Sequencing, Incubation, Ligation

    15) Product Images from ""

    Article Title:

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.284992

    Reaction of T4 DNA ligase with substrate 1 ( A ) and adenylylated substrate 1A ( B ) under single turnover conditions.  Each reaction was run with 500 n m  ligase and 100 n m  substrate in the standard ATP-free assay buffer. Ligase that was  > 95% adenylylated was used for  A , and
    Figure Legend Snippet: Reaction of T4 DNA ligase with substrate 1 ( A ) and adenylylated substrate 1A ( B ) under single turnover conditions. Each reaction was run with 500 n m ligase and 100 n m substrate in the standard ATP-free assay buffer. Ligase that was > 95% adenylylated was used for A , and

    Techniques Used:

    Pre-steady state reactions of 30 n m  (♦) and 50 n m  (■) T4 DNA ligase with 100 n m  substrate 1.  Reactions were run in the standard assay buffer. Each time point represents the average of three experiments, and the  error bars  represent one S.D. The  dashed lines  represent fits by simulation using the chemical rates determined from single turnover reaction of substrate  1 , literature values for Step 1 rates, and diffusion-limited binding of DNA and allowing the rate of product release ( k off ) and the amplitude ( a ) to vary. The best fit was obtained with  a  = 0.51 and  k off  = 0.58 s −1 .
    Figure Legend Snippet: Pre-steady state reactions of 30 n m (♦) and 50 n m (■) T4 DNA ligase with 100 n m substrate 1. Reactions were run in the standard assay buffer. Each time point represents the average of three experiments, and the error bars represent one S.D. The dashed lines represent fits by simulation using the chemical rates determined from single turnover reaction of substrate 1 , literature values for Step 1 rates, and diffusion-limited binding of DNA and allowing the rate of product release ( k off ) and the amplitude ( a ) to vary. The best fit was obtained with a = 0.51 and k off = 0.58 s −1 .

    Techniques Used: Diffusion-based Assay, Binding Assay

    Determination of  k cat  and  k cat / K m  for T4 DNA ligase and nicked substrates.  Shown is reaction of 1 n m  T4 DNA ligase with 1 n m  (○), 2 n m  (*), 5 n m  (×), 10 n m  (△), 20 n m  (♢), and 50 n m  (□) substrate  1  in standard assay buffer at 16 °C ( A ) and 1 n m  T4 DNA ligase (
    Figure Legend Snippet: Determination of k cat and k cat / K m for T4 DNA ligase and nicked substrates. Shown is reaction of 1 n m T4 DNA ligase with 1 n m (○), 2 n m (*), 5 n m (×), 10 n m (△), 20 n m (♢), and 50 n m (□) substrate 1 in standard assay buffer at 16 °C ( A ) and 1 n m T4 DNA ligase (

    Techniques Used:

    16) Product Images from ""

    Article Title:

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.284992

    Reaction of T4 DNA ligase with substrate 1 ( A ) and adenylylated substrate 1A ( B ) under single turnover conditions.  Each reaction was run with 500 n m  ligase and 100 n m  substrate in the standard ATP-free assay buffer. Ligase that was  > 95% adenylylated was used for  A , and
    Figure Legend Snippet: Reaction of T4 DNA ligase with substrate 1 ( A ) and adenylylated substrate 1A ( B ) under single turnover conditions. Each reaction was run with 500 n m ligase and 100 n m substrate in the standard ATP-free assay buffer. Ligase that was > 95% adenylylated was used for A , and

    Techniques Used:

    Pre-steady state reactions of 30 n m  (♦) and 50 n m  (■) T4 DNA ligase with 100 n m  substrate 1.  Reactions were run in the standard assay buffer. Each time point represents the average of three experiments, and the  error bars  represent one S.D. The  dashed lines  represent fits by simulation using the chemical rates determined from single turnover reaction of substrate  1 , literature values for Step 1 rates, and diffusion-limited binding of DNA and allowing the rate of product release ( k off ) and the amplitude ( a ) to vary. The best fit was obtained with  a  = 0.51 and  k off  = 0.58 s −1 .
    Figure Legend Snippet: Pre-steady state reactions of 30 n m (♦) and 50 n m (■) T4 DNA ligase with 100 n m substrate 1. Reactions were run in the standard assay buffer. Each time point represents the average of three experiments, and the error bars represent one S.D. The dashed lines represent fits by simulation using the chemical rates determined from single turnover reaction of substrate 1 , literature values for Step 1 rates, and diffusion-limited binding of DNA and allowing the rate of product release ( k off ) and the amplitude ( a ) to vary. The best fit was obtained with a = 0.51 and k off = 0.58 s −1 .

    Techniques Used: Diffusion-based Assay, Binding Assay

    Determination of  k cat  and  k cat / K m  for T4 DNA ligase and nicked substrates.  Shown is reaction of 1 n m  T4 DNA ligase with 1 n m  (○), 2 n m  (*), 5 n m  (×), 10 n m  (△), 20 n m  (♢), and 50 n m  (□) substrate  1  in standard assay buffer at 16 °C ( A ) and 1 n m  T4 DNA ligase (
    Figure Legend Snippet: Determination of k cat and k cat / K m for T4 DNA ligase and nicked substrates. Shown is reaction of 1 n m T4 DNA ligase with 1 n m (○), 2 n m (*), 5 n m (×), 10 n m (△), 20 n m (♢), and 50 n m (□) substrate 1 in standard assay buffer at 16 °C ( A ) and 1 n m T4 DNA ligase (

    Techniques Used:

    17) Product Images from ""

    Article Title:

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.284992

    Reaction of T4 DNA ligase with substrate 1 ( A ) and adenylylated substrate 1A ( B ) under single turnover conditions.  Each reaction was run with 500 n m  ligase and 100 n m  substrate in the standard ATP-free assay buffer. Ligase that was  > 95% adenylylated was used for  A , and
    Figure Legend Snippet: Reaction of T4 DNA ligase with substrate 1 ( A ) and adenylylated substrate 1A ( B ) under single turnover conditions. Each reaction was run with 500 n m ligase and 100 n m substrate in the standard ATP-free assay buffer. Ligase that was > 95% adenylylated was used for A , and

    Techniques Used:

    Pre-steady state reactions of 30 n m  (♦) and 50 n m  (■) T4 DNA ligase with 100 n m  substrate 1.  Reactions were run in the standard assay buffer. Each time point represents the average of three experiments, and the  error bars  represent one S.D. The  dashed lines  represent fits by simulation using the chemical rates determined from single turnover reaction of substrate  1 , literature values for Step 1 rates, and diffusion-limited binding of DNA and allowing the rate of product release ( k off ) and the amplitude ( a ) to vary. The best fit was obtained with  a  = 0.51 and  k off  = 0.58 s −1 .
    Figure Legend Snippet: Pre-steady state reactions of 30 n m (♦) and 50 n m (■) T4 DNA ligase with 100 n m substrate 1. Reactions were run in the standard assay buffer. Each time point represents the average of three experiments, and the error bars represent one S.D. The dashed lines represent fits by simulation using the chemical rates determined from single turnover reaction of substrate 1 , literature values for Step 1 rates, and diffusion-limited binding of DNA and allowing the rate of product release ( k off ) and the amplitude ( a ) to vary. The best fit was obtained with a = 0.51 and k off = 0.58 s −1 .

    Techniques Used: Diffusion-based Assay, Binding Assay

    Determination of  k cat  and  k cat / K m  for T4 DNA ligase and nicked substrates.  Shown is reaction of 1 n m  T4 DNA ligase with 1 n m  (○), 2 n m  (*), 5 n m  (×), 10 n m  (△), 20 n m  (♢), and 50 n m  (□) substrate  1  in standard assay buffer at 16 °C ( A ) and 1 n m  T4 DNA ligase (
    Figure Legend Snippet: Determination of k cat and k cat / K m for T4 DNA ligase and nicked substrates. Shown is reaction of 1 n m T4 DNA ligase with 1 n m (○), 2 n m (*), 5 n m (×), 10 n m (△), 20 n m (♢), and 50 n m (□) substrate 1 in standard assay buffer at 16 °C ( A ) and 1 n m T4 DNA ligase (

    Techniques Used:

    18) Product Images from "Probing transient protein-mediated DNA linkages using nanoconfinement"

    Article Title: Probing transient protein-mediated DNA linkages using nanoconfinement

    Journal:

    doi: 10.1063/1.4882775

    AFM images of DNA-DNA crossings. (a) Bare DNA (3.8 kbp). (b) and (c) DNA with T4 DNA ligase andATP. Solid arrows indicate higher crossings consistent with ligase binding, outlined arrows indicateshallower crossings consistent with bare DNA.
    Figure Legend Snippet: AFM images of DNA-DNA crossings. (a) Bare DNA (3.8 kbp). (b) and (c) DNA with T4 DNA ligase andATP. Solid arrows indicate higher crossings consistent with ligase binding, outlined arrows indicateshallower crossings consistent with bare DNA.

    Techniques Used: Binding Assay

    Mean aligned DNA molecule loop lengths as function of time for 22 molecules per dataset withtheir linear fits. Bare λ-DNA (blue), λ-DNA with T4 DNA ligase (green), and λ-DNA with T4 DNA ligaseand ATP (red).
    Figure Legend Snippet: Mean aligned DNA molecule loop lengths as function of time for 22 molecules per dataset withtheir linear fits. Bare λ-DNA (blue), λ-DNA with T4 DNA ligase (green), and λ-DNA with T4 DNA ligaseand ATP (red).

    Techniques Used:

    Histogram of end-to-end lengths of extended DNA molecules, bare λ-DNA (solid bars), λ-DNA with T4DNA ligase (gray bars), and λ-DNA with T4 DNA ligase and ATP (white bars). A Gaussian was fit toeach distribution to determine the
    Figure Legend Snippet: Histogram of end-to-end lengths of extended DNA molecules, bare λ-DNA (solid bars), λ-DNA with T4DNA ligase (gray bars), and λ-DNA with T4 DNA ligase and ATP (white bars). A Gaussian was fit toeach distribution to determine the

    Techniques Used:

    Histograms of heights of DNA-DNA crossings. (a) Bare DNA (N = 41). (b) DNA with T4 DNA ligase andATP (N = 174). The red dotted line corresponds to unoccupied crossings, the blue dashed line tooccupied crossings, and the
    Figure Legend Snippet: Histograms of heights of DNA-DNA crossings. (a) Bare DNA (N = 41). (b) DNA with T4 DNA ligase andATP (N = 174). The red dotted line corresponds to unoccupied crossings, the blue dashed line tooccupied crossings, and the

    Techniques Used:

    19) Product Images from "Probing transient protein-mediated DNA linkages using nanoconfinement"

    Article Title: Probing transient protein-mediated DNA linkages using nanoconfinement

    Journal:

    doi: 10.1063/1.4882775

    AFM images of DNA-DNA crossings. (a) Bare DNA (3.8 kbp). (b) and (c) DNA with T4 DNA ligase andATP. Solid arrows indicate higher crossings consistent with ligase binding, outlined arrows indicateshallower crossings consistent with bare DNA.
    Figure Legend Snippet: AFM images of DNA-DNA crossings. (a) Bare DNA (3.8 kbp). (b) and (c) DNA with T4 DNA ligase andATP. Solid arrows indicate higher crossings consistent with ligase binding, outlined arrows indicateshallower crossings consistent with bare DNA.

    Techniques Used: Binding Assay

    Mean aligned DNA molecule loop lengths as function of time for 22 molecules per dataset withtheir linear fits. Bare λ-DNA (blue), λ-DNA with T4 DNA ligase (green), and λ-DNA with T4 DNA ligaseand ATP (red).
    Figure Legend Snippet: Mean aligned DNA molecule loop lengths as function of time for 22 molecules per dataset withtheir linear fits. Bare λ-DNA (blue), λ-DNA with T4 DNA ligase (green), and λ-DNA with T4 DNA ligaseand ATP (red).

    Techniques Used:

    Histogram of end-to-end lengths of extended DNA molecules, bare λ-DNA (solid bars), λ-DNA with T4DNA ligase (gray bars), and λ-DNA with T4 DNA ligase and ATP (white bars). A Gaussian was fit toeach distribution to determine the
    Figure Legend Snippet: Histogram of end-to-end lengths of extended DNA molecules, bare λ-DNA (solid bars), λ-DNA with T4DNA ligase (gray bars), and λ-DNA with T4 DNA ligase and ATP (white bars). A Gaussian was fit toeach distribution to determine the

    Techniques Used:

    Histograms of heights of DNA-DNA crossings. (a) Bare DNA (N = 41). (b) DNA with T4 DNA ligase andATP (N = 174). The red dotted line corresponds to unoccupied crossings, the blue dashed line tooccupied crossings, and the
    Figure Legend Snippet: Histograms of heights of DNA-DNA crossings. (a) Bare DNA (N = 41). (b) DNA with T4 DNA ligase andATP (N = 174). The red dotted line corresponds to unoccupied crossings, the blue dashed line tooccupied crossings, and the

    Techniques Used:

    20) Product Images from "The yeast high mobility group protein HMO2, a subunit of the chromatin-remodeling complex INO80, binds DNA ends"

    Article Title: The yeast high mobility group protein HMO2, a subunit of the chromatin-remodeling complex INO80, binds DNA ends

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp695

    Interaction of HMO2 with plasmid DNA. ( A ,  B ) Agarose gel retardation of 100 ng plasmid DNA titrated with HMO2. (A) Reactions with supercoiled pGEM5. Lane 1, DNA only, lanes 2–7 with 1.0–6.0 μM HMO2. (B) Reactions with linearized pGEM5. Lane 1, DNA only, lanes 2–6 with 1.0–5.0 μM HMO2. ( C ) HMO2 supercoils relaxed DNA. Lane 1, 100 ng supercoiled pUC18 DNA. Lane 2, nicked pUC18. Lane 3, nicked pUC18 and T4 DNA ligase. Lanes 4–8, nicked DNA and T4 DNA ligase with 100, 500, 1000, 2000 and 3000 nM HMO2.
    Figure Legend Snippet: Interaction of HMO2 with plasmid DNA. ( A , B ) Agarose gel retardation of 100 ng plasmid DNA titrated with HMO2. (A) Reactions with supercoiled pGEM5. Lane 1, DNA only, lanes 2–7 with 1.0–6.0 μM HMO2. (B) Reactions with linearized pGEM5. Lane 1, DNA only, lanes 2–6 with 1.0–5.0 μM HMO2. ( C ) HMO2 supercoils relaxed DNA. Lane 1, 100 ng supercoiled pUC18 DNA. Lane 2, nicked pUC18. Lane 3, nicked pUC18 and T4 DNA ligase. Lanes 4–8, nicked DNA and T4 DNA ligase with 100, 500, 1000, 2000 and 3000 nM HMO2.

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis

    HMO2 prevents ligation of DNA by T4 DNA ligase. ( A ) DNA with overhangs (5′-TA extensions). ( B ) DNA with blunt ends. Lanes 1, 100 ng of DNA (∼4 nM, corresponding to ∼8 nM DNA ends). Lane 2, DNA and T4 DNA ligase. Lanes 3–8, DNA, T4 DNA ligase with 100, 500, 1000, 2000, 3000 and 4000 nM HMO2. Lane 9, DNA, T4 DNA ligase, 4000 nM HMO2 and exonuclease III.
    Figure Legend Snippet: HMO2 prevents ligation of DNA by T4 DNA ligase. ( A ) DNA with overhangs (5′-TA extensions). ( B ) DNA with blunt ends. Lanes 1, 100 ng of DNA (∼4 nM, corresponding to ∼8 nM DNA ends). Lane 2, DNA and T4 DNA ligase. Lanes 3–8, DNA, T4 DNA ligase with 100, 500, 1000, 2000, 3000 and 4000 nM HMO2. Lane 9, DNA, T4 DNA ligase, 4000 nM HMO2 and exonuclease III.

    Techniques Used: Ligation

    HMO1 promotes DNA end-joining, but does not protect DNA from exonucleolytic cleavage. ( A ) HMO1 can promote end-joining of pGEM5 DNA with 2-nt 5′ overhang in presence of T4 DNA ligase. Lane 1, 100 ng DNA only. Lane 2, DNA and T4 DNA ligase. Lanes 3–5, DNA, T4 DNA ligase, and 500, 1000 and 2000 nM HMO1, respectively. ( B ) HMO1 is unable to protect DNA with 2-nt 5′ overhangs from exonuclease III. Lane 1, 100 ng DNA only. Lane 2, DNA and exonuclease III. Lane 3, DNA and 500 nM HMO1. Lanes 4–6, DNA, exonuclease III, and 500, 1000 and 2000 nM HMO1, respectively.
    Figure Legend Snippet: HMO1 promotes DNA end-joining, but does not protect DNA from exonucleolytic cleavage. ( A ) HMO1 can promote end-joining of pGEM5 DNA with 2-nt 5′ overhang in presence of T4 DNA ligase. Lane 1, 100 ng DNA only. Lane 2, DNA and T4 DNA ligase. Lanes 3–5, DNA, T4 DNA ligase, and 500, 1000 and 2000 nM HMO1, respectively. ( B ) HMO1 is unable to protect DNA with 2-nt 5′ overhangs from exonuclease III. Lane 1, 100 ng DNA only. Lane 2, DNA and exonuclease III. Lane 3, DNA and 500 nM HMO1. Lanes 4–6, DNA, exonuclease III, and 500, 1000 and 2000 nM HMO1, respectively.

    Techniques Used:

    DNA protection by HMO2 depends on DNA length and sequence of DNA overhangs. ( A ) DNA with G+C-containing overhangs is not protected by HMO2. Lanes 1–4, DNA with 5′-CATG extensions (∼2 nM), lanes 5–8, DNA with 5′-TA extensions (∼4 nM). Lanes 1 and 5, DNA only. Lanes 2 and 6, DNA treated with exonuclease III for 1 h. Lanes 3 and 7, DNA and 2000 nM HMO2. Lanes 4 and 8, DNA with 2000 nM HMO2 incubated with exonuclease III for 1 h. Note in lane 8 the appearance of a product with lower mobility. Only the two largest fragments of BspHI-digested pET5a are shown in lanes 1–4. ( B ) Ligation of DNA with 5′-CATG extension (∼2 nM). Lane 1, DNA only. Lane 2, DNA and T4 DNA ligase. Lane 3, DNA, T4 DNA ligase and 2.5 µM HMO2. ( C ) Length dependence of DNA protection by HMO2. Lane 1, DNA with 4-nt 5′ overhangs. Lane 2, DNA treated with exonuclease III for 1 h. Lane 3, DNA and 2000 nM HMO2. Lane 4, DNA incubated with HMO2 and exonuclease III for 1 h. ( D ) HMO2 can end-join 105 bp DNA in presence of T4 DNA ligase. Lane 1, 100 fmol of 105 bp DNA. Lane 2, 105 bp DNA and T4 DNA ligase. Lanes 3–5, 105 bp DNA, T4 DNA ligase and 100, 250 and 500 nM HMO2. Lane 6, 105 bp DNA, T4 DNA ligase and 100 nM  B. subtilis  HU (HBsu). Lane 7, 105 bp DNA, T4 DNA ligase, 100 nM  B. subtilis  HU and exonuclease III. Lane 8, 105 bp DNA, T4 DNA ligase, 250 nM HMO2 and exonuclease III.
    Figure Legend Snippet: DNA protection by HMO2 depends on DNA length and sequence of DNA overhangs. ( A ) DNA with G+C-containing overhangs is not protected by HMO2. Lanes 1–4, DNA with 5′-CATG extensions (∼2 nM), lanes 5–8, DNA with 5′-TA extensions (∼4 nM). Lanes 1 and 5, DNA only. Lanes 2 and 6, DNA treated with exonuclease III for 1 h. Lanes 3 and 7, DNA and 2000 nM HMO2. Lanes 4 and 8, DNA with 2000 nM HMO2 incubated with exonuclease III for 1 h. Note in lane 8 the appearance of a product with lower mobility. Only the two largest fragments of BspHI-digested pET5a are shown in lanes 1–4. ( B ) Ligation of DNA with 5′-CATG extension (∼2 nM). Lane 1, DNA only. Lane 2, DNA and T4 DNA ligase. Lane 3, DNA, T4 DNA ligase and 2.5 µM HMO2. ( C ) Length dependence of DNA protection by HMO2. Lane 1, DNA with 4-nt 5′ overhangs. Lane 2, DNA treated with exonuclease III for 1 h. Lane 3, DNA and 2000 nM HMO2. Lane 4, DNA incubated with HMO2 and exonuclease III for 1 h. ( D ) HMO2 can end-join 105 bp DNA in presence of T4 DNA ligase. Lane 1, 100 fmol of 105 bp DNA. Lane 2, 105 bp DNA and T4 DNA ligase. Lanes 3–5, 105 bp DNA, T4 DNA ligase and 100, 250 and 500 nM HMO2. Lane 6, 105 bp DNA, T4 DNA ligase and 100 nM B. subtilis HU (HBsu). Lane 7, 105 bp DNA, T4 DNA ligase, 100 nM B. subtilis HU and exonuclease III. Lane 8, 105 bp DNA, T4 DNA ligase, 250 nM HMO2 and exonuclease III.

    Techniques Used: Sequencing, Incubation, Ligation

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    Article Snippet: The coding sequences of BRUSH and brush were then combined in a BsaI cut-ligation with modules containing the T7 promoter as well as the 5’UTR and 3’UTR sequences of β-globin mRNA (amplified from pEMHE). .. Plasmids were assembled in a 15 µl reaction containing 100 ng of each LI plasmid and backbone, 1.5 µl CutSmart buffer (NEB, Germany), 1.5 µl 10 mM ATP, 0.75 µl BsaI (NEB), 0.75 µl T4 ligase (NEB).

    Article Title: Genetic and epigenetic variations associated with adaptation to heterogeneous habitat conditions in a deciduous shrub. Genetic and epigenetic variations associated with adaptation to heterogeneous habitat conditions in a deciduous shrub
    Article Snippet: The reaction was incubated at 37°C for 2 hr and inactivated at 80°C for 20 min. Then, the product was combined with 48 μT4 DNA ligase (NEB), 1.4 μl 10 × T4 DNA ligase buffer, 1 μl EcoR I adapter (5 μM), and 1 μl Mse I adapter (50 μM) or 1 μl Hpa II/Msp I adapter (50 μM). .. The reaction was incubated at 37°C for 2 hr and inactivated at 80°C for 20 min. Then, the product was combined with 48 μT4 DNA ligase (NEB), 1.4 μl 10 × T4 DNA ligase buffer, 1 μl EcoR I adapter (5 μM), and 1 μl Mse I adapter (50 μM) or 1 μl Hpa II/Msp I adapter (50 μM).

    Article Title: Viral proteins as a potential driver of histone depletion in dinoflagellates
    Article Snippet: Sequencing libraries were constructed using 2 ng of DNA using a low-input protocol . .. Briefly, samples were end repaired (1X T4 DNA ligase buffer (NEB), 0.4 mM dNTP mix, 2.25 U T4 DNA polymerase (NEB), 0.75 U Klenow DNA polymerase (NEB), and 7.5 U of T4 polynucleotide kinase (NEB), incubated at room temperature for 30 min), A-tailed (1X NEB buffer 2, 0.4 mM dATP, and 3.75 U of Klenow (exo-) (NEB) incubated at 37 °C for 30 min), ligated to adapters (1X Quick DNA ligase buffer (NEB), 1 mM Illumina PE adapters, and 1600 U Quick DNA-ligase (NEB), incubated at room temperature for 1 h) and PCR amplified (1X NEBNext master mix (NEB) and 0.4 μM indexed primers (Illumina)) using 12 PCR cycles with a 65 °C annealing temperature and a 30 s extension time. .. DNA was purified between each step using two volumes of NucleoMag NGS DNA purification beads (Macherey–Nagel) except after adapter ligation and PCR amplification where 0.8 volumes were used to facilitate size selection.

    Reporter Assay:

    Article Title: Identification of nonsense-mediated mRNA decay pathway as a critical regulator of p53 isoform β
    Article Snippet: Paragraph title: p53 Reporter Assay ... The fragments were ligated using T4 DNA ligase (NEB); in-frame insertion was confirmed via diagnostic restriction enzyme digestion and sequencing.

    Mass Spectrometry:

    Article Title: Genetic and epigenetic variations associated with adaptation to heterogeneous habitat conditions in a deciduous shrub. Genetic and epigenetic variations associated with adaptation to heterogeneous habitat conditions in a deciduous shrub
    Article Snippet: Paragraph title: AFLP and MS‐AFLP protocol ... The reaction was incubated at 37°C for 2 hr and inactivated at 80°C for 20 min. Then, the product was combined with 48 μT4 DNA ligase (NEB), 1.4 μl 10 × T4 DNA ligase buffer, 1 μl EcoR I adapter (5 μM), and 1 μl Mse I adapter (50 μM) or 1 μl Hpa II/Msp I adapter (50 μM).

    Synthesized:

    Article Title: Multi-Platform Sequencing Approach Reveals a Novel Transcriptome Profile in Pseudorabies Virus
    Article Snippet: The direct RNA sequencing approach Libraries were prepared using the Direct RNA Sequencing Kit (SQK-RNA001; Oxford Nanopore Technologies) The first strand cDNA was synthesized by SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific) using an RT adapter with T10 nts. .. Adapters, supplied in the kit, were ligated using T4 DNA ligase (New England Biolabs).

    Construct:

    Article Title: dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression
    Article Snippet: Finally, the FOG1 epigenetic effector construct was Gibson assembled (New England Biolabs). .. Two, three or four monomer coding sequences were mixed with pFusA plasmid for Golden Gate Assembly cloning with BsaI and T4 DNA ligase (New England Biolabs).

    Article Title: An ApiAP2 member regulates expression of clonally variant genes of the human malaria parasite Plasmodium falciparum
    Article Snippet: Paragraph title: Plasmid constructs ... Ligation was done using T4 DNA ligase (NEB) and transformed into XL10-Gold ultracompetent cells (Agilent).

    Article Title: Hepatitis E Virus (HEV) egress: Role of BST2 (Tetherin) and interferon induced long non- coding RNA (lncRNA) BISPR
    Article Snippet: Final screening of colonies carrying dual gRNA ligated Cas9 constructs was done by Sanger sequencing using H1 primer. .. The left and right homology arms to be cloned at KpN1 and BamH1 sites of HR vector (Cat no: HR410PA-1, Systems Biosciences, Palo Alto, California, United States) were PCR amplified from region 17405497–17406190 and 17414509–17415316 (on chromosome 19) respectively from Huh7 genomic DNA using combination of Taq (Applied Biosystems, Foster City, California, United States) and Pfu DNA polymerase (Promega, Madison, Wisconsin, United States) in a 4:1 ratio, gel eluted, digested with KpN1 and BamH1 respectively and sequentially ligated into HRP410 vector using T4 DNA Ligase (NEB, Ipswich, Massachusetts, United Kingdom).

    Article Title: An activity-dependent proximity ligation platform for spatially resolved quantification of active enzymes in single cells
    Article Snippet: PAFAH2 (Origene, #RC200355) and ESD (Origene, #RC200533) constructs were digested using EcoR1-HF (New England BioLabs, #R3101S) and Fse1 (New England BioLabs, #R0588S), followed by heat inactivation. .. Backbone and insert were ligated using T4 DNA ligase (New England BioLabs, #M0202).

    Article Title: Identification of nonsense-mediated mRNA decay pathway as a critical regulator of p53 isoform β
    Article Snippet: Using the pCL-Neo β-globin WT Renilla construct we first digested with Xho1 restriction enzyme (NEB) and treated with T4 DNA polymerase (NEB) purified via PCR column and digested with Not1 restriction enzyme (NEB) for removal of the β-globin portion of the construct. cDNA from wild type HCT116 cells was used to PCR amplify our p53 isoform fragments using the Phusion Polymerase (Thermo Scientific). .. The fragments were ligated using T4 DNA ligase (NEB); in-frame insertion was confirmed via diagnostic restriction enzyme digestion and sequencing.

    Article Title: SALP, a new single-stranded DNA library preparation method especially useful for the high-throughput characterization of chromatin openness states
    Article Snippet: This method can be used to construct NGS library with the DNA fragments sheared by various methods including tagmentation, sonication and enzymatic digestion. .. This method constructs NGS library in several simple steps just dependent on two cheap routine enzymes, T4 DNA ligase and Taq polymerase. .. Combining with Tn5 tagmentation technique, this method can simply the NGS library construction procedure in five steps, including tagmentation, SSA ligation, elongation, T adaptor ligation, and index PCR amplification.

    Article Title: Small Non-coding RNA RyhB Mediates Persistence to Multiple Antibiotics and Stresses in Uropathogenic Escherichia coli by Reducing Cellular Metabolism
    Article Snippet: The arabinose-inducible plasmid pBAD202 was used to construct overexpression strains according to previous report (Ma et al., ). .. Genes were amplified with PCR primers, followed by digestion of both the PCR fragments and pBAD202 with the restriction enzymes Nco I and Eco RI (New England Biolabs, Ipswich, MA, USA) and ligation using the T4 DNA ligase (New England Biolabs).

    Article Title: A quantitative hypermorphic CNGC allele confers ectopic calcium flux and impairs cellular development
    Article Snippet: The same backbone was used to express the constructs for BiFC analysis, where LI Golden Gate B-C or D-E parts encoding for the N-terminal (VN) or C-terminal (VC) portions of mVenus ( ) were inserted. .. Plasmids were assembled in a 15 µl reaction containing 100 ng of each LI plasmid and backbone, 1.5 µl CutSmart buffer (NEB, Germany), 1.5 µl 10 mM ATP, 0.75 µl BsaI (NEB), 0.75 µl T4 ligase (NEB).

    Article Title: Viral proteins as a potential driver of histone depletion in dinoflagellates
    Article Snippet: Sequencing libraries were constructed using 2 ng of DNA using a low-input protocol . .. Briefly, samples were end repaired (1X T4 DNA ligase buffer (NEB), 0.4 mM dNTP mix, 2.25 U T4 DNA polymerase (NEB), 0.75 U Klenow DNA polymerase (NEB), and 7.5 U of T4 polynucleotide kinase (NEB), incubated at room temperature for 30 min), A-tailed (1X NEB buffer 2, 0.4 mM dATP, and 3.75 U of Klenow (exo-) (NEB) incubated at 37 °C for 30 min), ligated to adapters (1X Quick DNA ligase buffer (NEB), 1 mM Illumina PE adapters, and 1600 U Quick DNA-ligase (NEB), incubated at room temperature for 1 h) and PCR amplified (1X NEBNext master mix (NEB) and 0.4 μM indexed primers (Illumina)) using 12 PCR cycles with a 65 °C annealing temperature and a 30 s extension time.

    Electrophoresis:

    Article Title: An activity-dependent proximity ligation platform for spatially resolved quantification of active enzymes in single cells
    Article Snippet: Backbone and insert were ligated using T4 DNA ligase (New England BioLabs, #M0202). .. Backbone and insert were ligated using T4 DNA ligase (New England BioLabs, #M0202).

    Incubation:

    Article Title: A method for measuring the distribution of the shortest telomeres in cells and tissues
    Article Snippet: To ligate TeSLA-T (TeSLA-T 1–6) to each telomere overhang, 1000 units of T4 DNA ligase (New England Biolabs), 1 mM ATP, and 10−3 μM of TeSLA-Ts were added to a final volume of 20 μl in 1× CutSmart buffer (New England Biolabs) with 50 ng of isolated genomic DNA (without RE digestion). .. For adaptor ligation, 10 μl of the inactivated mixture was combined with 1000 units of T4 DNA ligase to a final volume of 20 μl in 1× CutSmart buffer with 1 mM ATP, 1 μM of AT adapter, and 1 μM of TA adapter.

    Article Title: An activity-dependent proximity ligation platform for spatially resolved quantification of active enzymes in single cells
    Article Snippet: Backbone and insert were ligated using T4 DNA ligase (New England BioLabs, #M0202). .. NEB 5-alpha competent Escherichia coli (high efficiency) cells (New England BioLabs, #C2987I) were transformed with the ligated plasmid.

    Article Title: A bacterial three-hybrid assay detects Escherichia coli Hfq–sRNA interactions in vivo
    Article Snippet: The PCR product was digested with DpnI (New England Biolabs) to remove template plasmid, then with NotI-HF and BamHI-HF (New England Biolabs), gel purified and ligated (T4 DNA ligase; New England Biolabs) into a pBRα vector cut with NotI-HF and BamHI-HF. .. For the primary screen, the pBRα-Hfq plasmid library was transformed into KB473 cells along with pKB989 (pACλCI-MS2CP ) and pKB912 (pCDF-MS2hp -OxyS) and plated on LB agar supplemented with inducers (0.2% arabinose and 1.5 μM IPTG), antibiotics (carbenicillin (50 μg/ml), chloramphenicol (25 μg/ml), kanamycin (50 μg/ml), and spectinomycin (100 μg/ml)) and indicators (Xgal (40 μg/ml) and phenylethyl-β-D-thiogalactopyranoside (125 μM; Gold Biotech)).

    Article Title: Genetic and epigenetic variations associated with adaptation to heterogeneous habitat conditions in a deciduous shrub. Genetic and epigenetic variations associated with adaptation to heterogeneous habitat conditions in a deciduous shrub
    Article Snippet: 150 ng) was combined with 10 μl double digestion mix containing 1 μl 10 × CutSmart Buffer (New England Biolabs, NEB), 2.5 μEco RI (NEB), 0.5 μMse I or 2.5 μHpa II or 2.5 μMsp I (NEB) in parallel reactions. .. The reaction was incubated at 37°C for 2 hr and inactivated at 80°C for 20 min. Then, the product was combined with 48 μT4 DNA ligase (NEB), 1.4 μl 10 × T4 DNA ligase buffer, 1 μl EcoR I adapter (5 μM), and 1 μl Mse I adapter (50 μM) or 1 μl Hpa II/Msp I adapter (50 μM). .. For the preselective amplification (PCR1), 2 μl ligation product was combined with 13 μl PCR1 reaction mix containing 0.7 μl preselective primers (5 μM) each, 0.6 μl dNTPs (TIANGEN, 2.5 mM each), 1.5 μl 10× buffer (TIAGEN), 0.75 U polymerase (TIAGEN), and 9.2 μl H2 O.

    Article Title: Viral proteins as a potential driver of histone depletion in dinoflagellates
    Article Snippet: Sequencing libraries were constructed using 2 ng of DNA using a low-input protocol . .. Briefly, samples were end repaired (1X T4 DNA ligase buffer (NEB), 0.4 mM dNTP mix, 2.25 U T4 DNA polymerase (NEB), 0.75 U Klenow DNA polymerase (NEB), and 7.5 U of T4 polynucleotide kinase (NEB), incubated at room temperature for 30 min), A-tailed (1X NEB buffer 2, 0.4 mM dATP, and 3.75 U of Klenow (exo-) (NEB) incubated at 37 °C for 30 min), ligated to adapters (1X Quick DNA ligase buffer (NEB), 1 mM Illumina PE adapters, and 1600 U Quick DNA-ligase (NEB), incubated at room temperature for 1 h) and PCR amplified (1X NEBNext master mix (NEB) and 0.4 μM indexed primers (Illumina)) using 12 PCR cycles with a 65 °C annealing temperature and a 30 s extension time. .. DNA was purified between each step using two volumes of NucleoMag NGS DNA purification beads (Macherey–Nagel) except after adapter ligation and PCR amplification where 0.8 volumes were used to facilitate size selection.

    Luciferase:

    Article Title: Identification of nonsense-mediated mRNA decay pathway as a critical regulator of p53 isoform β
    Article Snippet: The fragments were ligated using T4 DNA ligase (NEB); in-frame insertion was confirmed via diagnostic restriction enzyme digestion and sequencing. .. The fragments were ligated using T4 DNA ligase (NEB); in-frame insertion was confirmed via diagnostic restriction enzyme digestion and sequencing.

    In Silico:

    Article Title: GoldenPiCS: a Golden Gate-derived modular cloning system for applied synthetic biology in the yeast Pichia pastoris
    Article Snippet: Primer design and in silico cloning was performed using the CLC Main Workbench Version 7.7.3. .. Custom DNA oligonucleotides and gBlocks (from IDT, BE), restriction enzymes, T4 Ligase, Q5 polymerase (all from New England Biolabs, DE, or Fermentas, DE) and DNA cleanup kits (from Qiagen, DE, and Promega, DE) were used for routine cloning work.

    Expressing:

    Article Title: dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression
    Article Snippet: Paragraph title: Construction of dCas9 expression plasmids ... Two, three or four monomer coding sequences were mixed with pFusA plasmid for Golden Gate Assembly cloning with BsaI and T4 DNA ligase (New England Biolabs).

    Article Title: An ApiAP2 member regulates expression of clonally variant genes of the human malaria parasite Plasmodium falciparum
    Article Snippet: Ligation was done using T4 DNA ligase (NEB) and transformed into XL10-Gold ultracompetent cells (Agilent). .. PCR products were purified and ligated in the vector using T4 DNA ligase and the ligation product, transformed into chemically-competent DH5α E. coli cells.

    Article Title: An activity-dependent proximity ligation platform for spatially resolved quantification of active enzymes in single cells
    Article Snippet: Paragraph title: Lentiviral expression vector cloning ... Backbone and insert were ligated using T4 DNA ligase (New England BioLabs, #M0202).

    Article Title: Directly mining a fungal thermostable α-amylase from Chinese Nong-flavor liquor starter
    Article Snippet: Paragraph title: Gene cloning, expression and protein purification ... The resulting DNA fragments were ligated with T4 DNA ligase (New England Biolabs) and the ligated product was transformed into E. coli DH5a.

    Article Title: Small Non-coding RNA RyhB Mediates Persistence to Multiple Antibiotics and Stresses in Uropathogenic Escherichia coli by Reducing Cellular Metabolism
    Article Snippet: Genes were amplified with PCR primers, followed by digestion of both the PCR fragments and pBAD202 with the restriction enzymes Nco I and Eco RI (New England Biolabs, Ipswich, MA, USA) and ligation using the T4 DNA ligase (New England Biolabs). .. The deletion mutants and overexpression strains were verified by DNA sequencing.

    Article Title: A quantitative hypermorphic CNGC allele confers ectopic calcium flux and impairs cellular development
    Article Snippet: BRUSH and brush coding sequences were cloned for Xenopus expression with a custom Golden Gate cloning strategy using a modified backbone obtained from the Standard European Vector Architecture 2.0 database ( ). .. Plasmids were assembled in a 15 µl reaction containing 100 ng of each LI plasmid and backbone, 1.5 µl CutSmart buffer (NEB, Germany), 1.5 µl 10 mM ATP, 0.75 µl BsaI (NEB), 0.75 µl T4 ligase (NEB).

    Modification:

    Article Title: A quantitative hypermorphic CNGC allele confers ectopic calcium flux and impairs cellular development
    Article Snippet: BRUSH and brush coding sequences were cloned for Xenopus expression with a custom Golden Gate cloning strategy using a modified backbone obtained from the Standard European Vector Architecture 2.0 database ( ). .. Plasmids were assembled in a 15 µl reaction containing 100 ng of each LI plasmid and backbone, 1.5 µl CutSmart buffer (NEB, Germany), 1.5 µl 10 mM ATP, 0.75 µl BsaI (NEB), 0.75 µl T4 ligase (NEB).

    Transformation Assay:

    Article Title: An ApiAP2 member regulates expression of clonally variant genes of the human malaria parasite Plasmodium falciparum
    Article Snippet: Homology boxes were cloned into pCC1 plasmid by PCR amplification of the desired regions from 3D7 genomic DNA using specific primers flanked by appropriate restriction sites (list of primers, see Supplementary Table ). .. Ligation was done using T4 DNA ligase (NEB) and transformed into XL10-Gold ultracompetent cells (Agilent). .. For protein expression, we designed primers to amplify the N-terminal region of AP2-exp encompassing the AP2 DNA-binding domain into the pGEX-B vector (Pharmacia/GE Healthcare/Life Technologies).

    Article Title: An activity-dependent proximity ligation platform for spatially resolved quantification of active enzymes in single cells
    Article Snippet: Backbone and insert were ligated using T4 DNA ligase (New England BioLabs, #M0202). .. NEB 5-alpha competent Escherichia coli (high efficiency) cells (New England BioLabs, #C2987I) were transformed with the ligated plasmid.

    Article Title: Deep mutational scanning of S. pyogenes Cas9 reveals important functional domains
    Article Snippet: Six 20 µl golden gate cloning reactions were set up with 75 ng PCR product and 50 ng pUC-ProD-LacZ-T2 (Supplementary Fig. ) with 10 U Esp3I (Thermo Fisher Scientific) and 400 U T4 DNA Ligase (NEB) in 1X Ligase Buffer (NEB). .. Six 20 µl golden gate cloning reactions were set up with 75 ng PCR product and 50 ng pUC-ProD-LacZ-T2 (Supplementary Fig. ) with 10 U Esp3I (Thermo Fisher Scientific) and 400 U T4 DNA Ligase (NEB) in 1X Ligase Buffer (NEB).

    Article Title: A bacterial three-hybrid assay detects Escherichia coli Hfq–sRNA interactions in vivo
    Article Snippet: The PCR product was digested with DpnI (New England Biolabs) to remove template plasmid, then with NotI-HF and BamHI-HF (New England Biolabs), gel purified and ligated (T4 DNA ligase; New England Biolabs) into a pBRα vector cut with NotI-HF and BamHI-HF. .. Following ligation and transformation into NEB 5-α F’Iq cells (New England Biolabs), cells were grown as near-lawns on LB-carbenicillin plates and a miniprep was performed from resuspension of ∼30,000 colonies to yield the plasmid library.

    Article Title: Directly mining a fungal thermostable α-amylase from Chinese Nong-flavor liquor starter
    Article Snippet: The PCR products and plasmid pGAPZaA (Invitrogen) were digested with Kpn I and Xba I (New England Biolabs). .. The resulting DNA fragments were ligated with T4 DNA ligase (New England Biolabs) and the ligated product was transformed into E. coli DH5a. .. The transformants were selected on low salt LB (10 g/l tryptone, 5 g/l NaCl and 5 g/l yeast extract) agar plate supplemented with 25 μg/ml zeocin (Sangon Biotech, Shanghai).

    Over Expression:

    Article Title: An ApiAP2 member regulates expression of clonally variant genes of the human malaria parasite Plasmodium falciparum
    Article Snippet: Ligation was done using T4 DNA ligase (NEB) and transformed into XL10-Gold ultracompetent cells (Agilent). .. Sequencing of recombinant clones was done to confirm the correct ligation of the gene in frame with the glutathione S-transferase in the pGEX plasmid and BL21(DE3)RIL E. coli was transformed to obtain clones for protein expression.

    Article Title: Small Non-coding RNA RyhB Mediates Persistence to Multiple Antibiotics and Stresses in Uropathogenic Escherichia coli by Reducing Cellular Metabolism
    Article Snippet: Paragraph title: Construction of deletion mutants and overexpression strains ... Genes were amplified with PCR primers, followed by digestion of both the PCR fragments and pBAD202 with the restriction enzymes Nco I and Eco RI (New England Biolabs, Ipswich, MA, USA) and ligation using the T4 DNA ligase (New England Biolabs).

    Derivative Assay:

    Article Title: A quantitative hypermorphic CNGC allele confers ectopic calcium flux and impairs cellular development
    Article Snippet: The backbone (with flanking bacterial transcriptional terminators) was derived from pSEVA191 ( http://wwwuser.cnb.csic.es/~seva/ ) and was chosen to alleviate toxicity issues uncovered while cloning CNGC.IVA sequences into pUC-based Golden Gate backbones and pGEMHE ( ). .. Plasmids were assembled in a 15 µl reaction containing 100 ng of each LI plasmid and backbone, 1.5 µl CutSmart buffer (NEB, Germany), 1.5 µl 10 mM ATP, 0.75 µl BsaI (NEB), 0.75 µl T4 ligase (NEB).

    Flow Cytometry:

    Article Title: Multi-Platform Sequencing Approach Reveals a Novel Transcriptome Profile in Pseudorabies Virus
    Article Snippet: The Cap-selected mRNA sample was subjected to end repair and adapter ligation steps – as was described above – before loading on the Flow Cells. .. Adapters, supplied in the kit, were ligated using T4 DNA ligase (New England Biolabs).

    Ligation:

    Article Title: An ApiAP2 member regulates expression of clonally variant genes of the human malaria parasite Plasmodium falciparum
    Article Snippet: Homology boxes were cloned into pCC1 plasmid by PCR amplification of the desired regions from 3D7 genomic DNA using specific primers flanked by appropriate restriction sites (list of primers, see Supplementary Table ). .. Ligation was done using T4 DNA ligase (NEB) and transformed into XL10-Gold ultracompetent cells (Agilent). .. For protein expression, we designed primers to amplify the N-terminal region of AP2-exp encompassing the AP2 DNA-binding domain into the pGEX-B vector (Pharmacia/GE Healthcare/Life Technologies).

    Article Title: A method for measuring the distribution of the shortest telomeres in cells and tissues
    Article Snippet: Before starting the TeSLA procedure, we make stocks of short double-stranded 5′ AT and TA overhang adapters for ligation at genomic and subtelomeric regions. .. To ligate TeSLA-T (TeSLA-T 1–6) to each telomere overhang, 1000 units of T4 DNA ligase (New England Biolabs), 1 mM ATP, and 10−3 μM of TeSLA-Ts were added to a final volume of 20 μl in 1× CutSmart buffer (New England Biolabs) with 50 ng of isolated genomic DNA (without RE digestion).

    Article Title: Multi-Platform Sequencing Approach Reveals a Novel Transcriptome Profile in Pseudorabies Virus
    Article Snippet: The Cap-selected mRNA sample was subjected to end repair and adapter ligation steps – as was described above – before loading on the Flow Cells. .. Adapters, supplied in the kit, were ligated using T4 DNA ligase (New England Biolabs).

    Article Title: Small Non-coding RNA RyhB Mediates Persistence to Multiple Antibiotics and Stresses in Uropathogenic Escherichia coli by Reducing Cellular Metabolism
    Article Snippet: Primers (F: 5′-CATG CCATGGAAAAGCCAGCACCCGGC-3′ and R: 5′-CCCGGAATTCGCGATCAGGAAGACCCTCG-3′) used for the construction of the plasmid containing the RyhB gene were designed in this study. .. Genes were amplified with PCR primers, followed by digestion of both the PCR fragments and pBAD202 with the restriction enzymes Nco I and Eco RI (New England Biolabs, Ipswich, MA, USA) and ligation using the T4 DNA ligase (New England Biolabs). .. The new constructs along with the empty vector, pBAD202, were transformed into parent strain UTI89 for overexpression experiments.

    Magnetic Beads:

    Article Title: Multi-Platform Sequencing Approach Reveals a Novel Transcriptome Profile in Pseudorabies Virus
    Article Snippet: The cDNA sample was purified between each step using Agencourt AMPure XP magnetic beads (Beckman Coulter) and the library concentration was determined using a Qubit 2.0 Fluorometer through use of the Qubit (ds)DNA HS Assay Kit (Thermo Fisher Scientific). .. Adapters, supplied in the kit, were ligated using T4 DNA ligase (New England Biolabs).

    Cell Culture:

    Article Title: Directly mining a fungal thermostable α-amylase from Chinese Nong-flavor liquor starter
    Article Snippet: The resulting DNA fragments were ligated with T4 DNA ligase (New England Biolabs) and the ligated product was transformed into E. coli DH5a. .. The resulting DNA fragments were ligated with T4 DNA ligase (New England Biolabs) and the ligated product was transformed into E. coli DH5a.

    Generated:

    Article Title: Identification of nonsense-mediated mRNA decay pathway as a critical regulator of p53 isoform β
    Article Snippet: Constructs were generated using the NMD assay constructs as a backbone. .. The fragments were ligated using T4 DNA ligase (NEB); in-frame insertion was confirmed via diagnostic restriction enzyme digestion and sequencing.

    Article Title: A bacterial three-hybrid assay detects Escherichia coli Hfq–sRNA interactions in vivo
    Article Snippet: A mutant hfq library (pKB817, pBRα-Hfq) was generated first by 80 rounds of PCR amplification of the hfq portion of the plasmid using Taq DNA Polymerase (GoTaq Green Master Mix, Promega) and primers oKB1077 and oKB1078. .. The PCR product was digested with DpnI (New England Biolabs) to remove template plasmid, then with NotI-HF and BamHI-HF (New England Biolabs), gel purified and ligated (T4 DNA ligase; New England Biolabs) into a pBRα vector cut with NotI-HF and BamHI-HF.

    other:

    Article Title: Biomolecular computers with multiple restriction enzymes
    Article Snippet: The restriction enzymes Acu I, Bae I,Bbv I, Mbo II, Btgz I and T4 DNA ligase were obtained from New England Biolabs (Ipswich, MA, USA).

    DNA Sequencing:

    Article Title: An activity-dependent proximity ligation platform for spatially resolved quantification of active enzymes in single cells
    Article Snippet: Backbone and insert were ligated using T4 DNA ligase (New England BioLabs, #M0202). .. Transformed bacteria were plated on LB + Amp (100 µg/mL) agar plates and incubated at 37 °C overnight.

    Article Title: Directly mining a fungal thermostable α-amylase from Chinese Nong-flavor liquor starter
    Article Snippet: The resulting DNA fragments were ligated with T4 DNA ligase (New England Biolabs) and the ligated product was transformed into E. coli DH5a. .. Single colonies were inoculated into LB medium supplemented with same antibiotics, and cultured overnight.

    Article Title: Small Non-coding RNA RyhB Mediates Persistence to Multiple Antibiotics and Stresses in Uropathogenic Escherichia coli by Reducing Cellular Metabolism
    Article Snippet: Genes were amplified with PCR primers, followed by digestion of both the PCR fragments and pBAD202 with the restriction enzymes Nco I and Eco RI (New England Biolabs, Ipswich, MA, USA) and ligation using the T4 DNA ligase (New England Biolabs). .. The new constructs along with the empty vector, pBAD202, were transformed into parent strain UTI89 for overexpression experiments.

    Sequencing:

    Article Title: An ApiAP2 member regulates expression of clonally variant genes of the human malaria parasite Plasmodium falciparum
    Article Snippet: Ligation was done using T4 DNA ligase (NEB) and transformed into XL10-Gold ultracompetent cells (Agilent). .. PCR products were purified and ligated in the vector using T4 DNA ligase and the ligation product, transformed into chemically-competent DH5α E. coli cells.

    Article Title: Hepatitis E Virus (HEV) egress: Role of BST2 (Tetherin) and interferon induced long non- coding RNA (lncRNA) BISPR
    Article Snippet: Final screening of colonies carrying dual gRNA ligated Cas9 constructs was done by Sanger sequencing using H1 primer. .. The left and right homology arms to be cloned at KpN1 and BamH1 sites of HR vector (Cat no: HR410PA-1, Systems Biosciences, Palo Alto, California, United States) were PCR amplified from region 17405497–17406190 and 17414509–17415316 (on chromosome 19) respectively from Huh7 genomic DNA using combination of Taq (Applied Biosystems, Foster City, California, United States) and Pfu DNA polymerase (Promega, Madison, Wisconsin, United States) in a 4:1 ratio, gel eluted, digested with KpN1 and BamH1 respectively and sequentially ligated into HRP410 vector using T4 DNA Ligase (NEB, Ipswich, Massachusetts, United Kingdom).

    Article Title: An activity-dependent proximity ligation platform for spatially resolved quantification of active enzymes in single cells
    Article Snippet: Backbone and insert were ligated using T4 DNA ligase (New England BioLabs, #M0202). .. Transformed bacteria were plated on LB + Amp (100 µg/mL) agar plates and incubated at 37 °C overnight.

    Article Title: Deep mutational scanning of S. pyogenes Cas9 reveals important functional domains
    Article Snippet: A template plasmid, EF- RBS-SpCas9 (Supplementary Fig. ), was created with a ribosome binding site attached to a human codon optimized SpCas9 protein coding sequence (Supplementary Fig. ) and the appropriate Esp3I sites to be used for error-prone PCR by POE-PCR . .. Six 20 µl golden gate cloning reactions were set up with 75 ng PCR product and 50 ng pUC-ProD-LacZ-T2 (Supplementary Fig. ) with 10 U Esp3I (Thermo Fisher Scientific) and 400 U T4 DNA Ligase (NEB) in 1X Ligase Buffer (NEB).

    Article Title: Identification of nonsense-mediated mRNA decay pathway as a critical regulator of p53 isoform β
    Article Snippet: P53β forward primer: 5′- GACCAGACCAGCTTTCAAAAAGA-3′, p53γ forward primer: 5′-ATGCTACTTGACTTACGATGGTGTTACT-3′, p53 isoform reverse primer (Not1 digestion site): 5′aaagcggccgcTCAGTCTGAGTCAGGCCCTTCTG-3′. .. The fragments were ligated using T4 DNA ligase (NEB); in-frame insertion was confirmed via diagnostic restriction enzyme digestion and sequencing. .. The cells were transfected using Lipofectamine 2000 (Invitrogen) and harvested after 1.5 days for luciferase detection.

    Article Title: Multi-Platform Sequencing Approach Reveals a Novel Transcriptome Profile in Pseudorabies Virus
    Article Snippet: Paragraph title: Oxford Nanopore MinION Sequencing ... Adapters, supplied in the kit, were ligated using T4 DNA ligase (New England Biolabs).

    Article Title: Directly mining a fungal thermostable α-amylase from Chinese Nong-flavor liquor starter
    Article Snippet: The nucleotide sequence encoding the NFAmy13A was amplified with Mix (Green) using N3 cDNA as the template, with NFAf 5′ GGTACC GCGACTCCGGATGAGTGGAAAGCTCAG3′ and NFAr5′ TCTAGA CGCCGACGCACACAGACCACTCTTG3′ primers containing Kpn I and Xba I site, respectively. .. The resulting DNA fragments were ligated with T4 DNA ligase (New England Biolabs) and the ligated product was transformed into E. coli DH5a.

    Article Title: Viral proteins as a potential driver of histone depletion in dinoflagellates
    Article Snippet: Paragraph title: Sequencing and bioinformatic analysis ... Briefly, samples were end repaired (1X T4 DNA ligase buffer (NEB), 0.4 mM dNTP mix, 2.25 U T4 DNA polymerase (NEB), 0.75 U Klenow DNA polymerase (NEB), and 7.5 U of T4 polynucleotide kinase (NEB), incubated at room temperature for 30 min), A-tailed (1X NEB buffer 2, 0.4 mM dATP, and 3.75 U of Klenow (exo-) (NEB) incubated at 37 °C for 30 min), ligated to adapters (1X Quick DNA ligase buffer (NEB), 1 mM Illumina PE adapters, and 1600 U Quick DNA-ligase (NEB), incubated at room temperature for 1 h) and PCR amplified (1X NEBNext master mix (NEB) and 0.4 μM indexed primers (Illumina)) using 12 PCR cycles with a 65 °C annealing temperature and a 30 s extension time.

    Binding Assay:

    Article Title: Deep mutational scanning of S. pyogenes Cas9 reveals important functional domains
    Article Snippet: A template plasmid, EF- RBS-SpCas9 (Supplementary Fig. ), was created with a ribosome binding site attached to a human codon optimized SpCas9 protein coding sequence (Supplementary Fig. ) and the appropriate Esp3I sites to be used for error-prone PCR by POE-PCR . .. Six 20 µl golden gate cloning reactions were set up with 75 ng PCR product and 50 ng pUC-ProD-LacZ-T2 (Supplementary Fig. ) with 10 U Esp3I (Thermo Fisher Scientific) and 400 U T4 DNA Ligase (NEB) in 1X Ligase Buffer (NEB).

    RNA Sequencing Assay:

    Article Title: Multi-Platform Sequencing Approach Reveals a Novel Transcriptome Profile in Pseudorabies Virus
    Article Snippet: The direct RNA sequencing approach Libraries were prepared using the Direct RNA Sequencing Kit (SQK-RNA001; Oxford Nanopore Technologies) The first strand cDNA was synthesized by SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific) using an RT adapter with T10 nts. .. Adapters, supplied in the kit, were ligated using T4 DNA ligase (New England Biolabs).

    Methylation:

    Article Title: Genetic and epigenetic variations associated with adaptation to heterogeneous habitat conditions in a deciduous shrub. Genetic and epigenetic variations associated with adaptation to heterogeneous habitat conditions in a deciduous shrub
    Article Snippet: The protocol for MSAP was adapted from a standard AFLP (Vos et al. ), replacing the Mse I enzyme in two separate runs with the methylation‐sensitive enzymes Hpa II and Msp I using appropriate adaptors and primers. .. The reaction was incubated at 37°C for 2 hr and inactivated at 80°C for 20 min. Then, the product was combined with 48 μT4 DNA ligase (NEB), 1.4 μl 10 × T4 DNA ligase buffer, 1 μl EcoR I adapter (5 μM), and 1 μl Mse I adapter (50 μM) or 1 μl Hpa II/Msp I adapter (50 μM).

    Mutagenesis:

    Article Title: Deep mutational scanning of S. pyogenes Cas9 reveals important functional domains
    Article Snippet: Paragraph title: SpCas9 Mutant Library Cloning ... Six 20 µl golden gate cloning reactions were set up with 75 ng PCR product and 50 ng pUC-ProD-LacZ-T2 (Supplementary Fig. ) with 10 U Esp3I (Thermo Fisher Scientific) and 400 U T4 DNA Ligase (NEB) in 1X Ligase Buffer (NEB).

    Article Title: A bacterial three-hybrid assay detects Escherichia coli Hfq–sRNA interactions in vivo
    Article Snippet: A mutant hfq library (pKB817, pBRα-Hfq) was generated first by 80 rounds of PCR amplification of the hfq portion of the plasmid using Taq DNA Polymerase (GoTaq Green Master Mix, Promega) and primers oKB1077 and oKB1078. .. The PCR product was digested with DpnI (New England Biolabs) to remove template plasmid, then with NotI-HF and BamHI-HF (New England Biolabs), gel purified and ligated (T4 DNA ligase; New England Biolabs) into a pBRα vector cut with NotI-HF and BamHI-HF.

    Isolation:

    Article Title: A method for measuring the distribution of the shortest telomeres in cells and tissues
    Article Snippet: To make 40 μM AT and TA adapters, 40 μl of 100 μM TeSLA adapter short oligonucleotide (ONT) was mixed with 40 μl of 100 μM of TeSLA adapter TA and TeSLA adapter AT ONTs individually to make the final volume of 100 μl in 1× TSE buffer (10 mM Tris pH 8.0, 50 mM NaCl, 1 mM EDTA). .. To ligate TeSLA-T (TeSLA-T 1–6) to each telomere overhang, 1000 units of T4 DNA ligase (New England Biolabs), 1 mM ATP, and 10−3 μM of TeSLA-Ts were added to a final volume of 20 μl in 1× CutSmart buffer (New England Biolabs) with 50 ng of isolated genomic DNA (without RE digestion). .. The inactivated mixture including two units of Cvi AII in 10 μl 1× CutSmart buffer was incubated at 25 °C for 2 h to generate genomic DNA fragments with 5′ AT overhangs.

    Article Title: A bacterial three-hybrid assay detects Escherichia coli Hfq–sRNA interactions in vivo
    Article Snippet: The PCR product was digested with DpnI (New England Biolabs) to remove template plasmid, then with NotI-HF and BamHI-HF (New England Biolabs), gel purified and ligated (T4 DNA ligase; New England Biolabs) into a pBRα vector cut with NotI-HF and BamHI-HF. .. Plates were incubated overnight at 37°C, then at 4°C for an additional 4–8 h. Colonies that appeared white or pale from the primary screen were restreaked to confirm colony color.

    Bimolecular Fluorescence Complementation Assay:

    Article Title: A quantitative hypermorphic CNGC allele confers ectopic calcium flux and impairs cellular development
    Article Snippet: The same backbone was used to express the constructs for BiFC analysis, where LI Golden Gate B-C or D-E parts encoding for the N-terminal (VN) or C-terminal (VC) portions of mVenus ( ) were inserted. .. Plasmids were assembled in a 15 µl reaction containing 100 ng of each LI plasmid and backbone, 1.5 µl CutSmart buffer (NEB, Germany), 1.5 µl 10 mM ATP, 0.75 µl BsaI (NEB), 0.75 µl T4 ligase (NEB).

    Titration:

    Article Title: Deep mutational scanning of S. pyogenes Cas9 reveals important functional domains
    Article Snippet: Six 20 µl golden gate cloning reactions were set up with 75 ng PCR product and 50 ng pUC-ProD-LacZ-T2 (Supplementary Fig. ) with 10 U Esp3I (Thermo Fisher Scientific) and 400 U T4 DNA Ligase (NEB) in 1X Ligase Buffer (NEB). .. The purified ligations were transformed into five 25 µl aliquots of 10 G Elite electrocompetent cells (Lucigen), pulsed at 1800V, and recovered at 37 °C with shaking in 1 mL Recovery Media (Lucigen) for 1 hour.

    Protein Purification:

    Article Title: Directly mining a fungal thermostable α-amylase from Chinese Nong-flavor liquor starter
    Article Snippet: Paragraph title: Gene cloning, expression and protein purification ... The resulting DNA fragments were ligated with T4 DNA ligase (New England Biolabs) and the ligated product was transformed into E. coli DH5a.

    Polymerase Chain Reaction:

    Article Title: dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression
    Article Snippet: For array of two, three and four FOG1 domains to the N-terminus of dCas9, FOG1 monomer coding sequences were amplified separately by PCR introducing a GS linker between individual monomer coding sequences and the KpnI and FseI restriction sites at the beginning of first monomer and the end of the last monomer for each array. .. Two, three or four monomer coding sequences were mixed with pFusA plasmid for Golden Gate Assembly cloning with BsaI and T4 DNA ligase (New England Biolabs).

    Article Title: An ApiAP2 member regulates expression of clonally variant genes of the human malaria parasite Plasmodium falciparum
    Article Snippet: Homology boxes were cloned into pCC1 plasmid by PCR amplification of the desired regions from 3D7 genomic DNA using specific primers flanked by appropriate restriction sites (list of primers, see Supplementary Table ). .. Ligation was done using T4 DNA ligase (NEB) and transformed into XL10-Gold ultracompetent cells (Agilent).

    Article Title: A method for measuring the distribution of the shortest telomeres in cells and tissues
    Article Snippet: To ligate TeSLA-T (TeSLA-T 1–6) to each telomere overhang, 1000 units of T4 DNA ligase (New England Biolabs), 1 mM ATP, and 10−3 μM of TeSLA-Ts were added to a final volume of 20 μl in 1× CutSmart buffer (New England Biolabs) with 50 ng of isolated genomic DNA (without RE digestion). .. For adaptor ligation, 10 μl of the inactivated mixture was combined with 1000 units of T4 DNA ligase to a final volume of 20 μl in 1× CutSmart buffer with 1 mM ATP, 1 μM of AT adapter, and 1 μM of TA adapter.

    Article Title: Hepatitis E Virus (HEV) egress: Role of BST2 (Tetherin) and interferon induced long non- coding RNA (lncRNA) BISPR
    Article Snippet: Final screening of colonies carrying dual gRNA ligated Cas9 constructs was done by Sanger sequencing using H1 primer. .. The left and right homology arms to be cloned at KpN1 and BamH1 sites of HR vector (Cat no: HR410PA-1, Systems Biosciences, Palo Alto, California, United States) were PCR amplified from region 17405497–17406190 and 17414509–17415316 (on chromosome 19) respectively from Huh7 genomic DNA using combination of Taq (Applied Biosystems, Foster City, California, United States) and Pfu DNA polymerase (Promega, Madison, Wisconsin, United States) in a 4:1 ratio, gel eluted, digested with KpN1 and BamH1 respectively and sequentially ligated into HRP410 vector using T4 DNA Ligase (NEB, Ipswich, Massachusetts, United Kingdom). .. Sequence of primers used for amplification of homology arms is given in .

    Article Title: Deep mutational scanning of S. pyogenes Cas9 reveals important functional domains
    Article Snippet: The resulting amplicons were loaded on a gel and the appropriate amplicon at 4.4 kb was gel extracted and purified with a Qiagen gel extraction column. .. Six 20 µl golden gate cloning reactions were set up with 75 ng PCR product and 50 ng pUC-ProD-LacZ-T2 (Supplementary Fig. ) with 10 U Esp3I (Thermo Fisher Scientific) and 400 U T4 DNA Ligase (NEB) in 1X Ligase Buffer (NEB). .. The ligation reactions were put in a thermocycler running the program: 60 cycles of 37 °C for 5 minutes, 16 °C for 5 minutes, a final digestion at 37 °C for 30 minutes, and heat inactivation at 65 °C for 20 minutes.

    Article Title: Transcription coupled repair and biased insertion of human retrotransposon L1 in transcribed genes
    Article Snippet: Sheared plasmid DNA was primer extended using an oligo specific to the 3′ end of the synL1_neo rescue plasmid (3′_rescue_1: 5′ ATATATGAGTAACCTGAGGC 3′ or 3′_rescue_1_secondpA: 5′ GTGGGCATTCTGTCTTGTTC 3′). .. Duplexed T-linkers were ligated using 10 U T4 DNA ligase and PCR was performed using the primers: linker specific (5′ ACACTCTTTCCCTACACGACGCTCTTCCGATCT 3′) and 3′_rescue_1 (or 3′_rescue_1_secondpA) primer. .. PCR was carried out with these steps: initial denaturation at 94°, 20 cycles of 94° for 30s, 60° for 1 min, 72° for 1 min, and a final extension for 10 min at 72°.

    Article Title: Identification of nonsense-mediated mRNA decay pathway as a critical regulator of p53 isoform β
    Article Snippet: Using the pCL-Neo β-globin WT Renilla construct we first digested with Xho1 restriction enzyme (NEB) and treated with T4 DNA polymerase (NEB) purified via PCR column and digested with Not1 restriction enzyme (NEB) for removal of the β-globin portion of the construct. cDNA from wild type HCT116 cells was used to PCR amplify our p53 isoform fragments using the Phusion Polymerase (Thermo Scientific). .. The fragments were ligated using T4 DNA ligase (NEB); in-frame insertion was confirmed via diagnostic restriction enzyme digestion and sequencing.

    Article Title: A bacterial three-hybrid assay detects Escherichia coli Hfq–sRNA interactions in vivo
    Article Snippet: A mutant hfq library (pKB817, pBRα-Hfq) was generated first by 80 rounds of PCR amplification of the hfq portion of the plasmid using Taq DNA Polymerase (GoTaq Green Master Mix, Promega) and primers oKB1077 and oKB1078. .. The PCR product was digested with DpnI (New England Biolabs) to remove template plasmid, then with NotI-HF and BamHI-HF (New England Biolabs), gel purified and ligated (T4 DNA ligase; New England Biolabs) into a pBRα vector cut with NotI-HF and BamHI-HF. .. Following ligation and transformation into NEB 5-α F’Iq cells (New England Biolabs), cells were grown as near-lawns on LB-carbenicillin plates and a miniprep was performed from resuspension of ∼30,000 colonies to yield the plasmid library.

    Article Title: SALP, a new single-stranded DNA library preparation method especially useful for the high-throughput characterization of chromatin openness states
    Article Snippet: The Hind III-digested and sonicated gDNA was ligated with the SSA of 3N overhang and elongated with the same procedure as the tagmented DNA. .. Then the gDNA was ligated with a T adaptor in a 10-μL containing 1 μL of T adaptor (5 μM), 1× T4 DNA Ligase Reaction Buffer and 1 μL of T4 DNA ligase at 16 °C for 2 h. Finally, the gDNA was purified with 1.2× Ampure XP beads and amplified with different NEB index primers (Additional file : Table S1) in a 50-μL PCR reaction as described above. .. The PCR products were run with agarose gel and the gDNA fragments of 300–1000 bp were extracted with the QIAquick Gel Extraction Kit.

    Article Title: Directly mining a fungal thermostable α-amylase from Chinese Nong-flavor liquor starter
    Article Snippet: The PCR products and plasmid pGAPZaA (Invitrogen) were digested with Kpn I and Xba I (New England Biolabs). .. The resulting DNA fragments were ligated with T4 DNA ligase (New England Biolabs) and the ligated product was transformed into E. coli DH5a.

    Article Title: Small Non-coding RNA RyhB Mediates Persistence to Multiple Antibiotics and Stresses in Uropathogenic Escherichia coli by Reducing Cellular Metabolism
    Article Snippet: Primers (F: 5′-CATG CCATGGAAAAGCCAGCACCCGGC-3′ and R: 5′-CCCGGAATTCGCGATCAGGAAGACCCTCG-3′) used for the construction of the plasmid containing the RyhB gene were designed in this study. .. Genes were amplified with PCR primers, followed by digestion of both the PCR fragments and pBAD202 with the restriction enzymes Nco I and Eco RI (New England Biolabs, Ipswich, MA, USA) and ligation using the T4 DNA ligase (New England Biolabs). .. The new constructs along with the empty vector, pBAD202, were transformed into parent strain UTI89 for overexpression experiments.

    Article Title: Viral proteins as a potential driver of histone depletion in dinoflagellates
    Article Snippet: Sequencing libraries were constructed using 2 ng of DNA using a low-input protocol . .. Briefly, samples were end repaired (1X T4 DNA ligase buffer (NEB), 0.4 mM dNTP mix, 2.25 U T4 DNA polymerase (NEB), 0.75 U Klenow DNA polymerase (NEB), and 7.5 U of T4 polynucleotide kinase (NEB), incubated at room temperature for 30 min), A-tailed (1X NEB buffer 2, 0.4 mM dATP, and 3.75 U of Klenow (exo-) (NEB) incubated at 37 °C for 30 min), ligated to adapters (1X Quick DNA ligase buffer (NEB), 1 mM Illumina PE adapters, and 1600 U Quick DNA-ligase (NEB), incubated at room temperature for 1 h) and PCR amplified (1X NEBNext master mix (NEB) and 0.4 μM indexed primers (Illumina)) using 12 PCR cycles with a 65 °C annealing temperature and a 30 s extension time. .. DNA was purified between each step using two volumes of NucleoMag NGS DNA purification beads (Macherey–Nagel) except after adapter ligation and PCR amplification where 0.8 volumes were used to facilitate size selection.

    DNA HS Assay:

    Article Title: Multi-Platform Sequencing Approach Reveals a Novel Transcriptome Profile in Pseudorabies Virus
    Article Snippet: Adapters, supplied in the kit, were ligated using T4 DNA ligase (New England Biolabs). .. The RNA-DNA hybrid was purified between each step by using Agencourt AMPure XP magnetic beads (Beckman Coulter), treated with RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher Scientific).

    CRISPR:

    Article Title: Hepatitis E Virus (HEV) egress: Role of BST2 (Tetherin) and interferon induced long non- coding RNA (lncRNA) BISPR
    Article Snippet: Guide RNAs targeting human LncBISPR gene (chromosome 19, 17405686–17415736, Ncbi Accession no: NC_000019.10) were designed using CRISPR design tool ( crispr.mit.edu ; ). .. The left and right homology arms to be cloned at KpN1 and BamH1 sites of HR vector (Cat no: HR410PA-1, Systems Biosciences, Palo Alto, California, United States) were PCR amplified from region 17405497–17406190 and 17414509–17415316 (on chromosome 19) respectively from Huh7 genomic DNA using combination of Taq (Applied Biosystems, Foster City, California, United States) and Pfu DNA polymerase (Promega, Madison, Wisconsin, United States) in a 4:1 ratio, gel eluted, digested with KpN1 and BamH1 respectively and sequentially ligated into HRP410 vector using T4 DNA Ligase (NEB, Ipswich, Massachusetts, United Kingdom).

    Purification:

    Article Title: An ApiAP2 member regulates expression of clonally variant genes of the human malaria parasite Plasmodium falciparum
    Article Snippet: Ligation was done using T4 DNA ligase (NEB) and transformed into XL10-Gold ultracompetent cells (Agilent). .. Ligation was done using T4 DNA ligase (NEB) and transformed into XL10-Gold ultracompetent cells (Agilent).

    Article Title: Deep mutational scanning of S. pyogenes Cas9 reveals important functional domains
    Article Snippet: The resulting amplicons were loaded on a gel and the appropriate amplicon at 4.4 kb was gel extracted and purified with a Qiagen gel extraction column. .. Six 20 µl golden gate cloning reactions were set up with 75 ng PCR product and 50 ng pUC-ProD-LacZ-T2 (Supplementary Fig. ) with 10 U Esp3I (Thermo Fisher Scientific) and 400 U T4 DNA Ligase (NEB) in 1X Ligase Buffer (NEB).

    Article Title: Identification of nonsense-mediated mRNA decay pathway as a critical regulator of p53 isoform β
    Article Snippet: Using the pCL-Neo β-globin WT Renilla construct we first digested with Xho1 restriction enzyme (NEB) and treated with T4 DNA polymerase (NEB) purified via PCR column and digested with Not1 restriction enzyme (NEB) for removal of the β-globin portion of the construct. cDNA from wild type HCT116 cells was used to PCR amplify our p53 isoform fragments using the Phusion Polymerase (Thermo Scientific). .. The fragments were ligated using T4 DNA ligase (NEB); in-frame insertion was confirmed via diagnostic restriction enzyme digestion and sequencing.

    Article Title: A bacterial three-hybrid assay detects Escherichia coli Hfq–sRNA interactions in vivo
    Article Snippet: A mutant hfq library (pKB817, pBRα-Hfq) was generated first by 80 rounds of PCR amplification of the hfq portion of the plasmid using Taq DNA Polymerase (GoTaq Green Master Mix, Promega) and primers oKB1077 and oKB1078. .. The PCR product was digested with DpnI (New England Biolabs) to remove template plasmid, then with NotI-HF and BamHI-HF (New England Biolabs), gel purified and ligated (T4 DNA ligase; New England Biolabs) into a pBRα vector cut with NotI-HF and BamHI-HF. .. Following ligation and transformation into NEB 5-α F’Iq cells (New England Biolabs), cells were grown as near-lawns on LB-carbenicillin plates and a miniprep was performed from resuspension of ∼30,000 colonies to yield the plasmid library.

    Article Title: Multi-Platform Sequencing Approach Reveals a Novel Transcriptome Profile in Pseudorabies Virus
    Article Snippet: The cDNA sample was purified between each step using Agencourt AMPure XP magnetic beads (Beckman Coulter) and the library concentration was determined using a Qubit 2.0 Fluorometer through use of the Qubit (ds)DNA HS Assay Kit (Thermo Fisher Scientific). .. Adapters, supplied in the kit, were ligated using T4 DNA ligase (New England Biolabs).

    Article Title: SALP, a new single-stranded DNA library preparation method especially useful for the high-throughput characterization of chromatin openness states
    Article Snippet: The Hind III-digested and sonicated gDNA was ligated with the SSA of 3N overhang and elongated with the same procedure as the tagmented DNA. .. Then the gDNA was ligated with a T adaptor in a 10-μL containing 1 μL of T adaptor (5 μM), 1× T4 DNA Ligase Reaction Buffer and 1 μL of T4 DNA ligase at 16 °C for 2 h. Finally, the gDNA was purified with 1.2× Ampure XP beads and amplified with different NEB index primers (Additional file : Table S1) in a 50-μL PCR reaction as described above. .. The PCR products were run with agarose gel and the gDNA fragments of 300–1000 bp were extracted with the QIAquick Gel Extraction Kit.

    Plasmid Preparation:

    Article Title: dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression
    Article Snippet: Amplification primers are listed in . .. Two, three or four monomer coding sequences were mixed with pFusA plasmid for Golden Gate Assembly cloning with BsaI and T4 DNA ligase (New England Biolabs). .. DNA fragments of arrays of two, three and four FOG1 domains were digested with KpnI and FseI and ligated into the KpnI/FseI digested dCas9 plasmid.

    Article Title: An ApiAP2 member regulates expression of clonally variant genes of the human malaria parasite Plasmodium falciparum
    Article Snippet: Paragraph title: Plasmid constructs ... Ligation was done using T4 DNA ligase (NEB) and transformed into XL10-Gold ultracompetent cells (Agilent).

    Article Title: Hepatitis E Virus (HEV) egress: Role of BST2 (Tetherin) and interferon induced long non- coding RNA (lncRNA) BISPR
    Article Snippet: Final screening of colonies carrying dual gRNA ligated Cas9 constructs was done by Sanger sequencing using H1 primer. .. The left and right homology arms to be cloned at KpN1 and BamH1 sites of HR vector (Cat no: HR410PA-1, Systems Biosciences, Palo Alto, California, United States) were PCR amplified from region 17405497–17406190 and 17414509–17415316 (on chromosome 19) respectively from Huh7 genomic DNA using combination of Taq (Applied Biosystems, Foster City, California, United States) and Pfu DNA polymerase (Promega, Madison, Wisconsin, United States) in a 4:1 ratio, gel eluted, digested with KpN1 and BamH1 respectively and sequentially ligated into HRP410 vector using T4 DNA Ligase (NEB, Ipswich, Massachusetts, United Kingdom). .. Sequence of primers used for amplification of homology arms is given in .

    Article Title: An activity-dependent proximity ligation platform for spatially resolved quantification of active enzymes in single cells
    Article Snippet: Paragraph title: Lentiviral expression vector cloning ... Backbone and insert were ligated using T4 DNA ligase (New England BioLabs, #M0202).

    Article Title: Deep mutational scanning of S. pyogenes Cas9 reveals important functional domains
    Article Snippet: A template plasmid, EF- RBS-SpCas9 (Supplementary Fig. ), was created with a ribosome binding site attached to a human codon optimized SpCas9 protein coding sequence (Supplementary Fig. ) and the appropriate Esp3I sites to be used for error-prone PCR by POE-PCR . .. Six 20 µl golden gate cloning reactions were set up with 75 ng PCR product and 50 ng pUC-ProD-LacZ-T2 (Supplementary Fig. ) with 10 U Esp3I (Thermo Fisher Scientific) and 400 U T4 DNA Ligase (NEB) in 1X Ligase Buffer (NEB).

    Article Title: A bacterial three-hybrid assay detects Escherichia coli Hfq–sRNA interactions in vivo
    Article Snippet: A mutant hfq library (pKB817, pBRα-Hfq) was generated first by 80 rounds of PCR amplification of the hfq portion of the plasmid using Taq DNA Polymerase (GoTaq Green Master Mix, Promega) and primers oKB1077 and oKB1078. .. The PCR product was digested with DpnI (New England Biolabs) to remove template plasmid, then with NotI-HF and BamHI-HF (New England Biolabs), gel purified and ligated (T4 DNA ligase; New England Biolabs) into a pBRα vector cut with NotI-HF and BamHI-HF. .. Following ligation and transformation into NEB 5-α F’Iq cells (New England Biolabs), cells were grown as near-lawns on LB-carbenicillin plates and a miniprep was performed from resuspension of ∼30,000 colonies to yield the plasmid library.

    Article Title: Directly mining a fungal thermostable α-amylase from Chinese Nong-flavor liquor starter
    Article Snippet: The PCR products and plasmid pGAPZaA (Invitrogen) were digested with Kpn I and Xba I (New England Biolabs). .. The resulting DNA fragments were ligated with T4 DNA ligase (New England Biolabs) and the ligated product was transformed into E. coli DH5a.

    Article Title: Small Non-coding RNA RyhB Mediates Persistence to Multiple Antibiotics and Stresses in Uropathogenic Escherichia coli by Reducing Cellular Metabolism
    Article Snippet: Primers (F: 5′-CATG CCATGGAAAAGCCAGCACCCGGC-3′ and R: 5′-CCCGGAATTCGCGATCAGGAAGACCCTCG-3′) used for the construction of the plasmid containing the RyhB gene were designed in this study. .. Genes were amplified with PCR primers, followed by digestion of both the PCR fragments and pBAD202 with the restriction enzymes Nco I and Eco RI (New England Biolabs, Ipswich, MA, USA) and ligation using the T4 DNA ligase (New England Biolabs).

    Article Title: A quantitative hypermorphic CNGC allele confers ectopic calcium flux and impairs cellular development
    Article Snippet: The same backbone was used to express the constructs for BiFC analysis, where LI Golden Gate B-C or D-E parts encoding for the N-terminal (VN) or C-terminal (VC) portions of mVenus ( ) were inserted. .. Plasmids were assembled in a 15 µl reaction containing 100 ng of each LI plasmid and backbone, 1.5 µl CutSmart buffer (NEB, Germany), 1.5 µl 10 mM ATP, 0.75 µl BsaI (NEB), 0.75 µl T4 ligase (NEB). .. The reaction was then cycled 6 times (10 min at 37°C, 10 min 16°C) in a PCR machine, followed by incubation at 37°C (10 min) and 65°C (20 min).

    Software:

    Article Title: Multi-Platform Sequencing Approach Reveals a Novel Transcriptome Profile in Pseudorabies Virus
    Article Snippet: Adapters, supplied in the kit, were ligated using T4 DNA ligase (New England Biolabs). .. Libraries were loaded on R9.4 SpotON Flow Cells.

    Recombinant:

    Article Title: An ApiAP2 member regulates expression of clonally variant genes of the human malaria parasite Plasmodium falciparum
    Article Snippet: Ligation was done using T4 DNA ligase (NEB) and transformed into XL10-Gold ultracompetent cells (Agilent). .. PCR products were purified and ligated in the vector using T4 DNA ligase and the ligation product, transformed into chemically-competent DH5α E. coli cells.

    Article Title: Directly mining a fungal thermostable α-amylase from Chinese Nong-flavor liquor starter
    Article Snippet: The resulting DNA fragments were ligated with T4 DNA ligase (New England Biolabs) and the ligated product was transformed into E. coli DH5a. .. Plasmids were extracted (Qiagen mini-prep kit) from the cultures, and the inserts were verified by DNA sequencing (TsingKe, Chengdu, China).

    Agarose Gel Electrophoresis:

    Article Title: Viral proteins as a potential driver of histone depletion in dinoflagellates
    Article Snippet: Briefly, samples were end repaired (1X T4 DNA ligase buffer (NEB), 0.4 mM dNTP mix, 2.25 U T4 DNA polymerase (NEB), 0.75 U Klenow DNA polymerase (NEB), and 7.5 U of T4 polynucleotide kinase (NEB), incubated at room temperature for 30 min), A-tailed (1X NEB buffer 2, 0.4 mM dATP, and 3.75 U of Klenow (exo-) (NEB) incubated at 37 °C for 30 min), ligated to adapters (1X Quick DNA ligase buffer (NEB), 1 mM Illumina PE adapters, and 1600 U Quick DNA-ligase (NEB), incubated at room temperature for 1 h) and PCR amplified (1X NEBNext master mix (NEB) and 0.4 μM indexed primers (Illumina)) using 12 PCR cycles with a 65 °C annealing temperature and a 30 s extension time. .. Briefly, samples were end repaired (1X T4 DNA ligase buffer (NEB), 0.4 mM dNTP mix, 2.25 U T4 DNA polymerase (NEB), 0.75 U Klenow DNA polymerase (NEB), and 7.5 U of T4 polynucleotide kinase (NEB), incubated at room temperature for 30 min), A-tailed (1X NEB buffer 2, 0.4 mM dATP, and 3.75 U of Klenow (exo-) (NEB) incubated at 37 °C for 30 min), ligated to adapters (1X Quick DNA ligase buffer (NEB), 1 mM Illumina PE adapters, and 1600 U Quick DNA-ligase (NEB), incubated at room temperature for 1 h) and PCR amplified (1X NEBNext master mix (NEB) and 0.4 μM indexed primers (Illumina)) using 12 PCR cycles with a 65 °C annealing temperature and a 30 s extension time.

    Knock-Out:

    Article Title: Small Non-coding RNA RyhB Mediates Persistence to Multiple Antibiotics and Stresses in Uropathogenic Escherichia coli by Reducing Cellular Metabolism
    Article Snippet: Primers used to amplify all knockout-DNA fragments and verify the correct constructs by polymerase chain reaction (PCR) are shown in Supplementary Tables , . .. Genes were amplified with PCR primers, followed by digestion of both the PCR fragments and pBAD202 with the restriction enzymes Nco I and Eco RI (New England Biolabs, Ipswich, MA, USA) and ligation using the T4 DNA ligase (New England Biolabs).

    Next-Generation Sequencing:

    Article Title: SALP, a new single-stranded DNA library preparation method especially useful for the high-throughput characterization of chromatin openness states
    Article Snippet: This method can be used to construct NGS library with the DNA fragments sheared by various methods including tagmentation, sonication and enzymatic digestion. .. This method constructs NGS library in several simple steps just dependent on two cheap routine enzymes, T4 DNA ligase and Taq polymerase. .. Combining with Tn5 tagmentation technique, this method can simply the NGS library construction procedure in five steps, including tagmentation, SSA ligation, elongation, T adaptor ligation, and index PCR amplification.

    Spectrophotometry:

    Article Title: Genetic and epigenetic variations associated with adaptation to heterogeneous habitat conditions in a deciduous shrub. Genetic and epigenetic variations associated with adaptation to heterogeneous habitat conditions in a deciduous shrub
    Article Snippet: DNA was quantified with both 0.8% agarose gels and microscopic spectrophotometry. .. The reaction was incubated at 37°C for 2 hr and inactivated at 80°C for 20 min. Then, the product was combined with 48 μT4 DNA ligase (NEB), 1.4 μl 10 × T4 DNA ligase buffer, 1 μl EcoR I adapter (5 μM), and 1 μl Mse I adapter (50 μM) or 1 μl Hpa II/Msp I adapter (50 μM).

    Concentration Assay:

    Article Title: Multi-Platform Sequencing Approach Reveals a Novel Transcriptome Profile in Pseudorabies Virus
    Article Snippet: The cDNA sample was purified between each step using Agencourt AMPure XP magnetic beads (Beckman Coulter) and the library concentration was determined using a Qubit 2.0 Fluorometer through use of the Qubit (ds)DNA HS Assay Kit (Thermo Fisher Scientific). .. Adapters, supplied in the kit, were ligated using T4 DNA ligase (New England Biolabs).

    Gel Extraction:

    Article Title: Deep mutational scanning of S. pyogenes Cas9 reveals important functional domains
    Article Snippet: The resulting amplicons were loaded on a gel and the appropriate amplicon at 4.4 kb was gel extracted and purified with a Qiagen gel extraction column. .. Six 20 µl golden gate cloning reactions were set up with 75 ng PCR product and 50 ng pUC-ProD-LacZ-T2 (Supplementary Fig. ) with 10 U Esp3I (Thermo Fisher Scientific) and 400 U T4 DNA Ligase (NEB) in 1X Ligase Buffer (NEB).

    Blocking Assay:

    Article Title: Hepatitis E Virus (HEV) egress: Role of BST2 (Tetherin) and interferon induced long non- coding RNA (lncRNA) BISPR
    Article Snippet: Both gRNAs ( ) and U6 block (Systems Biosciences, Palo Alto, California, United States) were used as template to perform fusion PCR and generate ~450 bp H1-gRNA1-U6-gRNA2 amplicon ( ) using Precision XTM Cas9 SmartNuclease system (CAS740A). .. The left and right homology arms to be cloned at KpN1 and BamH1 sites of HR vector (Cat no: HR410PA-1, Systems Biosciences, Palo Alto, California, United States) were PCR amplified from region 17405497–17406190 and 17414509–17415316 (on chromosome 19) respectively from Huh7 genomic DNA using combination of Taq (Applied Biosystems, Foster City, California, United States) and Pfu DNA polymerase (Promega, Madison, Wisconsin, United States) in a 4:1 ratio, gel eluted, digested with KpN1 and BamH1 respectively and sequentially ligated into HRP410 vector using T4 DNA Ligase (NEB, Ipswich, Massachusetts, United Kingdom).

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  • 99
    New England Biolabs t4 dna ligase ligation protocol
    Effect of Inhibiting dsDNA on Enzyme Self-Adenylylation Rate. The determined rates for self-adenylylation of an uninhibited reaction, 2.5 μM T4 DNA ligase (red) and 2.5 μM T4 DNA ligase and inhibited reactions 2.5 μM DNA (blue) and 10 μM DNA (green). The reactions were fit to a single exponential equation (  Eq 6 ) to determine the reaction rate. The uninhibited reaction was determined to have a single turnover rate of 20 s -1  ± 2 s -1 . While the 2.5 μM inhibited reaction had a single turnover rate of 2.8 s -1  ± 0.5 s -1 and the 10 μM inhibited reaction had a single turnover rate of 1.0 s -1  ± 1 s -1 . All reactions were performed a minimum of three times at 16°C. Error reported is the standard error for the replicates.
    T4 Dna Ligase Ligation Protocol, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase ligation protocol/product/New England Biolabs
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase ligation protocol - by Bioz Stars, 2019-12
    99/100 stars
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    99
    New England Biolabs e coli dna gyrase
    Three representative time records (offset) showing plectonemes formed by the insertion of negative supercoils by E. coli <t>gyrase.</t> Normal or DAP-substituted <t>DNA</t> tethers were stretched with 0.3 pN of tension in the presence of E. coli gyrase and ATP. One
    E Coli Dna Gyrase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli dna gyrase/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    e coli dna gyrase - by Bioz Stars, 2019-12
    99/100 stars
      Buy from Supplier


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    Effect of Inhibiting dsDNA on Enzyme Self-Adenylylation Rate. The determined rates for self-adenylylation of an uninhibited reaction, 2.5 μM T4 DNA ligase (red) and 2.5 μM T4 DNA ligase and inhibited reactions 2.5 μM DNA (blue) and 10 μM DNA (green). The reactions were fit to a single exponential equation (  Eq 6 ) to determine the reaction rate. The uninhibited reaction was determined to have a single turnover rate of 20 s -1  ± 2 s -1 . While the 2.5 μM inhibited reaction had a single turnover rate of 2.8 s -1  ± 0.5 s -1 and the 10 μM inhibited reaction had a single turnover rate of 1.0 s -1  ± 1 s -1 . All reactions were performed a minimum of three times at 16°C. Error reported is the standard error for the replicates.

    Journal: PLoS ONE

    Article Title: The Inhibitory Effect of Non-Substrate and Substrate DNA on the Ligation and Self-Adenylylation Reactions Catalyzed by T4 DNA Ligase

    doi: 10.1371/journal.pone.0150802

    Figure Lengend Snippet: Effect of Inhibiting dsDNA on Enzyme Self-Adenylylation Rate. The determined rates for self-adenylylation of an uninhibited reaction, 2.5 μM T4 DNA ligase (red) and 2.5 μM T4 DNA ligase and inhibited reactions 2.5 μM DNA (blue) and 10 μM DNA (green). The reactions were fit to a single exponential equation ( Eq 6 ) to determine the reaction rate. The uninhibited reaction was determined to have a single turnover rate of 20 s -1 ± 2 s -1 . While the 2.5 μM inhibited reaction had a single turnover rate of 2.8 s -1 ± 0.5 s -1 and the 10 μM inhibited reaction had a single turnover rate of 1.0 s -1 ± 1 s -1 . All reactions were performed a minimum of three times at 16°C. Error reported is the standard error for the replicates.

    Article Snippet: DsDNA inhibition likely also has an effect in commonly used molecular biology protocols, for example, the maximal recommended DNA concentration utilized in the ligation step for Next Generation Sequencing library preparation is ~20 ng/μL (NEB Ultra II), while the recommended DNA concentration in a standard sticky-end ligation is 4.38 ng/μL (NEB T4 DNA ligase ligation protocol).

    Techniques:

    T4 DNA Ligase Reaction Model. Modified reaction pathway to include the newly observed reactions in the previously described DNA ligation pathway that are inhibited by the presence of non-nicked dsDNA.  A . Non-nicked dsDNA can bind to the deadenylylated form of the enzyme inhibition formation of the adenylylated form of the enzyme.  B . Non-nicked dsDNA binds to the Lig-AMP form, preventing complexation with its preferred ds-nDNA substrate.

    Journal: PLoS ONE

    Article Title: The Inhibitory Effect of Non-Substrate and Substrate DNA on the Ligation and Self-Adenylylation Reactions Catalyzed by T4 DNA Ligase

    doi: 10.1371/journal.pone.0150802

    Figure Lengend Snippet: T4 DNA Ligase Reaction Model. Modified reaction pathway to include the newly observed reactions in the previously described DNA ligation pathway that are inhibited by the presence of non-nicked dsDNA. A . Non-nicked dsDNA can bind to the deadenylylated form of the enzyme inhibition formation of the adenylylated form of the enzyme. B . Non-nicked dsDNA binds to the Lig-AMP form, preventing complexation with its preferred ds-nDNA substrate.

    Article Snippet: DsDNA inhibition likely also has an effect in commonly used molecular biology protocols, for example, the maximal recommended DNA concentration utilized in the ligation step for Next Generation Sequencing library preparation is ~20 ng/μL (NEB Ultra II), while the recommended DNA concentration in a standard sticky-end ligation is 4.38 ng/μL (NEB T4 DNA ligase ligation protocol).

    Techniques: Modification, DNA Ligation, Enzyme Inhibition Assay

    Competition for ds-nDNA-Binding by dsDNA. Lane one contains 4 nM of the 75mer-ds-nDNA substrate alone, lanes 2–6 show shifting of the 4 nM substrate into a completely bound state as the concentration of T4 DNA ligase is increased from 100 nM– 1000 nM. Lanes 7–11 are of a titration of increasing concentrations of the unlabeled I-75-dsDNA oligo into a reaction containing 4 nM labeled nicked substrate and 1000 nM T4 DNA ligase. EMSA reactions were all performed and electrophoresed at room temperature (22°C).

    Journal: PLoS ONE

    Article Title: The Inhibitory Effect of Non-Substrate and Substrate DNA on the Ligation and Self-Adenylylation Reactions Catalyzed by T4 DNA Ligase

    doi: 10.1371/journal.pone.0150802

    Figure Lengend Snippet: Competition for ds-nDNA-Binding by dsDNA. Lane one contains 4 nM of the 75mer-ds-nDNA substrate alone, lanes 2–6 show shifting of the 4 nM substrate into a completely bound state as the concentration of T4 DNA ligase is increased from 100 nM– 1000 nM. Lanes 7–11 are of a titration of increasing concentrations of the unlabeled I-75-dsDNA oligo into a reaction containing 4 nM labeled nicked substrate and 1000 nM T4 DNA ligase. EMSA reactions were all performed and electrophoresed at room temperature (22°C).

    Article Snippet: DsDNA inhibition likely also has an effect in commonly used molecular biology protocols, for example, the maximal recommended DNA concentration utilized in the ligation step for Next Generation Sequencing library preparation is ~20 ng/μL (NEB Ultra II), while the recommended DNA concentration in a standard sticky-end ligation is 4.38 ng/μL (NEB T4 DNA ligase ligation protocol).

    Techniques: Binding Assay, Concentration Assay, Titration, Labeling

    Various Inhibitors Effects on Rate of Nick Sealing. Various concentrations of dsDNA substrates were utilized as potential inhibitors of the T4 DNA ligase steady state ligation reaction on 20 nM of the 75mer-ds-nDNA substrate. All reactions were performed in the presence of 25 pM of T4 DNA ligase, a minimum of three times at 16°C. Error reported is the standard deviation for the replicates.

    Journal: PLoS ONE

    Article Title: The Inhibitory Effect of Non-Substrate and Substrate DNA on the Ligation and Self-Adenylylation Reactions Catalyzed by T4 DNA Ligase

    doi: 10.1371/journal.pone.0150802

    Figure Lengend Snippet: Various Inhibitors Effects on Rate of Nick Sealing. Various concentrations of dsDNA substrates were utilized as potential inhibitors of the T4 DNA ligase steady state ligation reaction on 20 nM of the 75mer-ds-nDNA substrate. All reactions were performed in the presence of 25 pM of T4 DNA ligase, a minimum of three times at 16°C. Error reported is the standard deviation for the replicates.

    Article Snippet: DsDNA inhibition likely also has an effect in commonly used molecular biology protocols, for example, the maximal recommended DNA concentration utilized in the ligation step for Next Generation Sequencing library preparation is ~20 ng/μL (NEB Ultra II), while the recommended DNA concentration in a standard sticky-end ligation is 4.38 ng/μL (NEB T4 DNA ligase ligation protocol).

    Techniques: Ligation, Standard Deviation

    k cat /K m  Curve for T4 DNA Ligase. The data was obtained through titration of increasing concentrations of a 75mer-ds-nDNA substrate, reacted at 16°C to determine initial reaction rates. T4 DNA ligase concentrations used were 25 pM– 100 pM. The initial rates were plotted against their respective substrate concentrations and fit by:  A . a classical uncompetitive substrate inhibition model (  Eq 2 ), where k cat  and K m  Values of 0.44 s -1  ± 0.3 s -1  and 4 nM ± 1 nM respectively, were determined. The K i  value for substrate inhibition was calculated to be 590 nM ± 170 nM.  B . A competitive substrate inhibition for a Bi-Bi Ping-Pong mechanism (  Eq 3 ) k cat  and K m  values of 0.48 s -1  ± 0.3 s -1  and 4 nM ± 1 nM respectively, were determined. The K i  value for substrate inhibition was calculated to be 54 nM ± 15 nM. All data points are the average of at least three independent experiments, and the error reported is the standard deviation for the replicates.

    Journal: PLoS ONE

    Article Title: The Inhibitory Effect of Non-Substrate and Substrate DNA on the Ligation and Self-Adenylylation Reactions Catalyzed by T4 DNA Ligase

    doi: 10.1371/journal.pone.0150802

    Figure Lengend Snippet: k cat /K m Curve for T4 DNA Ligase. The data was obtained through titration of increasing concentrations of a 75mer-ds-nDNA substrate, reacted at 16°C to determine initial reaction rates. T4 DNA ligase concentrations used were 25 pM– 100 pM. The initial rates were plotted against their respective substrate concentrations and fit by: A . a classical uncompetitive substrate inhibition model ( Eq 2 ), where k cat and K m Values of 0.44 s -1 ± 0.3 s -1 and 4 nM ± 1 nM respectively, were determined. The K i value for substrate inhibition was calculated to be 590 nM ± 170 nM. B . A competitive substrate inhibition for a Bi-Bi Ping-Pong mechanism ( Eq 3 ) k cat and K m values of 0.48 s -1 ± 0.3 s -1 and 4 nM ± 1 nM respectively, were determined. The K i value for substrate inhibition was calculated to be 54 nM ± 15 nM. All data points are the average of at least three independent experiments, and the error reported is the standard deviation for the replicates.

    Article Snippet: DsDNA inhibition likely also has an effect in commonly used molecular biology protocols, for example, the maximal recommended DNA concentration utilized in the ligation step for Next Generation Sequencing library preparation is ~20 ng/μL (NEB Ultra II), while the recommended DNA concentration in a standard sticky-end ligation is 4.38 ng/μL (NEB T4 DNA ligase ligation protocol).

    Techniques: Titration, Inhibition, Standard Deviation

    AFM images of DNA-DNA crossings. (a) Bare DNA (3.8 kbp). (b) and (c) DNA with T4 DNA ligase andATP. Solid arrows indicate higher crossings consistent with ligase binding, outlined arrows indicateshallower crossings consistent with bare DNA.

    Journal:

    Article Title: Probing transient protein-mediated DNA linkages using nanoconfinement

    doi: 10.1063/1.4882775

    Figure Lengend Snippet: AFM images of DNA-DNA crossings. (a) Bare DNA (3.8 kbp). (b) and (c) DNA with T4 DNA ligase andATP. Solid arrows indicate higher crossings consistent with ligase binding, outlined arrows indicateshallower crossings consistent with bare DNA.

    Article Snippet: We find thatλ-DNA contracted in the presence of T4 DNA ligase, from 14.4 μ m for bare DNA to 12.5 μ m with T4 DNA ligase.

    Techniques: Binding Assay

    Mean aligned DNA molecule loop lengths as function of time for 22 molecules per dataset withtheir linear fits. Bare λ-DNA (blue), λ-DNA with T4 DNA ligase (green), and λ-DNA with T4 DNA ligaseand ATP (red).

    Journal:

    Article Title: Probing transient protein-mediated DNA linkages using nanoconfinement

    doi: 10.1063/1.4882775

    Figure Lengend Snippet: Mean aligned DNA molecule loop lengths as function of time for 22 molecules per dataset withtheir linear fits. Bare λ-DNA (blue), λ-DNA with T4 DNA ligase (green), and λ-DNA with T4 DNA ligaseand ATP (red).

    Article Snippet: We find thatλ-DNA contracted in the presence of T4 DNA ligase, from 14.4 μ m for bare DNA to 12.5 μ m with T4 DNA ligase.

    Techniques:

    Histogram of end-to-end lengths of extended DNA molecules, bare λ-DNA (solid bars), λ-DNA with T4DNA ligase (gray bars), and λ-DNA with T4 DNA ligase and ATP (white bars). A Gaussian was fit toeach distribution to determine the

    Journal:

    Article Title: Probing transient protein-mediated DNA linkages using nanoconfinement

    doi: 10.1063/1.4882775

    Figure Lengend Snippet: Histogram of end-to-end lengths of extended DNA molecules, bare λ-DNA (solid bars), λ-DNA with T4DNA ligase (gray bars), and λ-DNA with T4 DNA ligase and ATP (white bars). A Gaussian was fit toeach distribution to determine the

    Article Snippet: We find thatλ-DNA contracted in the presence of T4 DNA ligase, from 14.4 μ m for bare DNA to 12.5 μ m with T4 DNA ligase.

    Techniques:

    Histograms of heights of DNA-DNA crossings. (a) Bare DNA (N = 41). (b) DNA with T4 DNA ligase andATP (N = 174). The red dotted line corresponds to unoccupied crossings, the blue dashed line tooccupied crossings, and the

    Journal:

    Article Title: Probing transient protein-mediated DNA linkages using nanoconfinement

    doi: 10.1063/1.4882775

    Figure Lengend Snippet: Histograms of heights of DNA-DNA crossings. (a) Bare DNA (N = 41). (b) DNA with T4 DNA ligase andATP (N = 174). The red dotted line corresponds to unoccupied crossings, the blue dashed line tooccupied crossings, and the

    Article Snippet: We find thatλ-DNA contracted in the presence of T4 DNA ligase, from 14.4 μ m for bare DNA to 12.5 μ m with T4 DNA ligase.

    Techniques:

    Three representative time records (offset) showing plectonemes formed by the insertion of negative supercoils by E. coli gyrase. Normal or DAP-substituted DNA tethers were stretched with 0.3 pN of tension in the presence of E. coli gyrase and ATP. One

    Journal:

    Article Title: E. coli gyrase fails to negatively supercoil diaminopurine-substituted DNA

    doi: 10.1016/j.jmb.2015.04.006

    Figure Lengend Snippet: Three representative time records (offset) showing plectonemes formed by the insertion of negative supercoils by E. coli gyrase. Normal or DAP-substituted DNA tethers were stretched with 0.3 pN of tension in the presence of E. coli gyrase and ATP. One

    Article Snippet: E. coli DNA gyrase (NEB) at varying concentrations (184 – 920 nM) was incubated with 40 nM of Cy5-labeled, 160 bp double stranded DNA for 30 min. in 35 mM Tris-HCl pH 7.5, 5 mM MgCl2 , 24 mM KCl, 2mM DTT and 10% glycerol at room temperature.

    Techniques:

    Decreased E. coli gyrase binding to DAP DNA. Equal amounts of normal or DAP DNA were incubated with increasing E. coli ). Average percentages of binding to gyrase at different

    Journal:

    Article Title: E. coli gyrase fails to negatively supercoil diaminopurine-substituted DNA

    doi: 10.1016/j.jmb.2015.04.006

    Figure Lengend Snippet: Decreased E. coli gyrase binding to DAP DNA. Equal amounts of normal or DAP DNA were incubated with increasing E. coli ). Average percentages of binding to gyrase at different

    Article Snippet: E. coli DNA gyrase (NEB) at varying concentrations (184 – 920 nM) was incubated with 40 nM of Cy5-labeled, 160 bp double stranded DNA for 30 min. in 35 mM Tris-HCl pH 7.5, 5 mM MgCl2 , 24 mM KCl, 2mM DTT and 10% glycerol at room temperature.

    Techniques: Binding Assay, Incubation

    Decreased gyrase wrapping of DAP DNA. (a) A schematic of the equilibrium between wrapped and unwrapped states of gyrase with no ATP present shows that the extension of the DNA tether switches between two levels. (b and c) Raw data (dots) and 10 second

    Journal:

    Article Title: E. coli gyrase fails to negatively supercoil diaminopurine-substituted DNA

    doi: 10.1016/j.jmb.2015.04.006

    Figure Lengend Snippet: Decreased gyrase wrapping of DAP DNA. (a) A schematic of the equilibrium between wrapped and unwrapped states of gyrase with no ATP present shows that the extension of the DNA tether switches between two levels. (b and c) Raw data (dots) and 10 second

    Article Snippet: E. coli DNA gyrase (NEB) at varying concentrations (184 – 920 nM) was incubated with 40 nM of Cy5-labeled, 160 bp double stranded DNA for 30 min. in 35 mM Tris-HCl pH 7.5, 5 mM MgCl2 , 24 mM KCl, 2mM DTT and 10% glycerol at room temperature.

    Techniques:

    The lifetimes of the wrapped states of gyrase for DAP (red circles) and normal (blue crosses) DNA under 0.4 pN of tension were measured and the fraction greater than or equal to different time intervals was plotted. The number of measured pauses was 154

    Journal:

    Article Title: E. coli gyrase fails to negatively supercoil diaminopurine-substituted DNA

    doi: 10.1016/j.jmb.2015.04.006

    Figure Lengend Snippet: The lifetimes of the wrapped states of gyrase for DAP (red circles) and normal (blue crosses) DNA under 0.4 pN of tension were measured and the fraction greater than or equal to different time intervals was plotted. The number of measured pauses was 154

    Article Snippet: E. coli DNA gyrase (NEB) at varying concentrations (184 – 920 nM) was incubated with 40 nM of Cy5-labeled, 160 bp double stranded DNA for 30 min. in 35 mM Tris-HCl pH 7.5, 5 mM MgCl2 , 24 mM KCl, 2mM DTT and 10% glycerol at room temperature.

    Techniques:

    Gyrase relaxes DAP DNA more slowly. (a) Three offset, representative records of gyrase-catalyzed relaxations of normal (left) and DAP DNA (right) under 0.6 pN tension and with 1 mM ATP. The raw data (dots) and 0.5 s moving averages (solid lines) show

    Journal:

    Article Title: E. coli gyrase fails to negatively supercoil diaminopurine-substituted DNA

    doi: 10.1016/j.jmb.2015.04.006

    Figure Lengend Snippet: Gyrase relaxes DAP DNA more slowly. (a) Three offset, representative records of gyrase-catalyzed relaxations of normal (left) and DAP DNA (right) under 0.6 pN tension and with 1 mM ATP. The raw data (dots) and 0.5 s moving averages (solid lines) show

    Article Snippet: E. coli DNA gyrase (NEB) at varying concentrations (184 – 920 nM) was incubated with 40 nM of Cy5-labeled, 160 bp double stranded DNA for 30 min. in 35 mM Tris-HCl pH 7.5, 5 mM MgCl2 , 24 mM KCl, 2mM DTT and 10% glycerol at room temperature.

    Techniques: