phage t4 dna ligase (New England Biolabs)
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Name:
T4 DNA Ligase
Description:
T4 DNA Ligase 100 000 units
Catalog Number:
m0202l
Price:
256
Size:
100 000 units
Category:
DNA Ligases
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Structured Review
New England Biolabs
phage t4 dna ligase
T4 DNA Ligase 100 000 units
https://www.bioz.com/result/phage t4 dna ligase/product/New England Biolabs
Average 95 stars, based on 3482 article reviews
Price from $9.99 to $1999.99
T4 DNA Ligase 100 000 units
https://www.bioz.com/result/phage t4 dna ligase/product/New England Biolabs
Average 95 stars, based on 3482 article reviews
Price from $9.99 to $1999.99
phage t4 dna ligase - by Bioz Stars,
2020-02
95/100 stars
Related Products / Commonly Used Together
Images
1) Product Images from "Quantitative disclosure of DNA knot chirality by high-resolution 2D-gel electrophoresis"
Article Title: Quantitative disclosure of DNA knot chirality by high-resolution 2D-gel electrophoresis
Journal: Nucleic Acids Research
doi: 10.1093/nar/gkz015
Figure Legend Snippet: Test of the electrophoresis procedure that discerns DNA knot chirality. ( A ) A linear 4.4-kb DNA fragment was circularized in free solution with T4 DNA ligase to produce a small fraction of molecules containing a trefoil knot. Negative supercoils were subsequently introduced by incubating the circularized DNA with topoisomerase I in presence of 250 μg/ml chloroquine. ( B ) The gel-blot shows the DNA products after high resolution 2D-gel electrophoresis carried out in 0.9% agarose gel (40 × 23 cm) in TBE. The first gel dimension (top to bottom) was run at 80 V for 70 h in TBE (89 mM Tris-borate, pH 8.3, 2 mM EDTA). The second gel dimension (left to right) was run at 120 V for 10 h in TBE containing 0.65 μg/ml of chloroquine. Lk, linking number topoisomers. N, nicked unknotted circles. L, linear DNA. The enlarged gel section shows the signal of Lk topoisomers of unknotted molecules (Kn# 0) and of molecules containing either a positive- or negative-noded trefoil knot (Kn# 3). ( C ) Probability of the two chiral forms of the trefoil knot.
Techniques Used: Electrophoresis, Western Blot, Two-Dimensional Gel Electrophoresis, Agarose Gel Electrophoresis
2) Product Images from "Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles"
Article Title: Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles
Journal: BMC Molecular Biology
doi: 10.1186/s12867-017-0083-2
Figure Legend Snippet: Flowchart of MSD-library preparation. Genomic DNA (100 ng) was digested with 10 units of the primary restriction enzyme Sbf I for 1 h and then ligated with 0.5 nmol Adaptor A using 400 units of T4 DNA ligase for 2 h. The treated sample was then digested with 100 units of the non-methylation-sensitive restriction enzyme Msp I (100 units) followed by ligation of the ends of the DNA fragment with Adaptor B. The ligated DNA fragments were then digested with 50 units of Hpa II for 1 h. Owing to the methylation sensitivity of Hap II, only DNA fragments with a methylated CpG retained Adaptor B, which was removed from all other fragments. The DNA fragments were then subjected to Pre-PCR using specific primers for Adaptor A and Adaptor B. Fragments that did not contain Adaptor B at this stage were not amplified. The Pre-PCR amplicons (MSD library) were then amplified as a subpopulation by selective-PCR with 6-carboxyfluorescein (6-FAM)-labelled selective-PCR primers. Finally, the selective-PCR products were electrophoresed with a capillary sequencer and separated by length
Techniques Used: Methylation, Ligation, Polymerase Chain Reaction, Amplification
3) Product Images from "Mutational phospho-mimicry reveals a regulatory role for the XRCC4 and XLF C-terminal tails in modulating DNA bridging during classical non-homologous end joining"
Article Title: Mutational phospho-mimicry reveals a regulatory role for the XRCC4 and XLF C-terminal tails in modulating DNA bridging during classical non-homologous end joining
Journal: eLife
doi: 10.7554/eLife.22900
Figure Legend Snippet: T4 DNA ligase assay using a 2.7 kb DNA fragment with cohesive (left) or blunt ends (right) at three different protein equimolar concentrations. ( A ) 2 μM each, ( B ) 1 μM each, and ( C ) 0.5 μM each. Ligation products were deproteinized and resolved by agarose gel electrophoresis followed by DNA detection by ethidium bromide staining. NC = nicked circle, CCC = covalently closed circle. DOI: http://dx.doi.org/10.7554/eLife.22900.006
Techniques Used: Ligation, Agarose Gel Electrophoresis, Staining, Countercurrent Chromatography
Figure Legend Snippet: Blocking XRCC4 and XLF phosphorylation sites enhances DNA bridging; phospho-mimicking mutations abate DNA bridging. All combinations of XRCC4 variants with XLF variants tested in ability to stimulate T4 DNA ligase cohesive end ligation ( A ) or blunt end ligation ( B ). Ligation products were deproteinized and resolved by agarose gel electrophoresis followed by detection by ethidium bromide staining. DOI: http://dx.doi.org/10.7554/eLife.22900.014
Techniques Used: Blocking Assay, Ligation, Agarose Gel Electrophoresis, Staining
4) Product Images from "Synapsis of Recombination Signal Sequences Located in cis and DNA Underwinding in V(D)J Recombination"
Article Title: Synapsis of Recombination Signal Sequences Located in cis and DNA Underwinding in V(D)J Recombination
Journal:
doi: 10.1128/MCB.24.19.8727-8744.2004
Figure Legend Snippet: Kinetics of RAG-mediated synapsis of cis RSSs. Complete ligation reactions were performed by adding a mixture containing 200 U of T4 DNA ligase, 12.5 nM GST-RAG2, and 6 nM MBP-RAG1-D708A to a solution containing 3 nM IS95 DNA in the presence of 2.5 mM
Techniques Used: Ligation
5) Product Images from "Patch cloning method for multiple site-directed and saturation mutagenesis"
Article Title: Patch cloning method for multiple site-directed and saturation mutagenesis
Journal: BMC Biotechnology
doi: 10.1186/1472-6750-13-91
Figure Legend Snippet: Schematic illustration of the multiple patch cloning procedure. DNA fragments are amplified by polymerase chain reaction using two sets of oligo-DNA primers (shown in red and blue). The star on the primer indicates the site of mismatch. The resultant DNA fragments and digested vector DNA containing 16 bp homologous regions (shown in yellow) were assembled at 37°C by T5 exonuclease, Klenow fragment and T4 DNA ligase.
Techniques Used: Clone Assay, Amplification, Polymerase Chain Reaction, Plasmid Preparation
6) Product Images from "7,8-dihydro-8-oxoadenine, a highly mutagenic adduct, is repaired by Escherichia coli and human mismatch-specific uracil/thymine-DNA glycosylases"
Article Title: 7,8-dihydro-8-oxoadenine, a highly mutagenic adduct, is repaired by Escherichia coli and human mismatch-specific uracil/thymine-DNA glycosylases
Journal: Nucleic Acids Research
doi: 10.1093/nar/gks1149
![In vitro reconstitution of the BER pathway using 8oxoA•T duplex DNA substrate. 5 nM 40 mer 8oxoA•T duplex was incubated in the presence of 20 nM hTDG, 5 nM APE1, 2 nM FEN1, 0.1 U POL-β and 5 nM T4 DNA ligase in buffer containing 20 µCi of [α- 32 P]dATP, 50 µM dNTPs, 50 mM HEPES–KOH (pH 7.6), 30 mM NaCl, 0.1 mg/ml BSA, 2 mM DTT, 2 mM ATP and 3 mM MgCl 2 for 5 and 30 min at 37°C. Lane 1, 30 min in the absence of hTDG and T4 DNA ligase; lane 2, 5 min in the absence of T4 DNA ligase; lane 3, same as 2, but 30 min; lane 4, 30 min in the presence of all proteins. For details see ‘Materials and Methods’ section.](https://storage.googleapis.com/bioz_article_images/PMC3553953/gks1149f3p.jpg "... nM FEN1, 0.1 U POL-β and 5 nM T4 DNA ligase in buffer containing 20 µCi of ...")
Figure Legend Snippet: In vitro reconstitution of the BER pathway using 8oxoA•T duplex DNA substrate. 5 nM 40 mer 8oxoA•T duplex was incubated in the presence of 20 nM hTDG, 5 nM APE1, 2 nM FEN1, 0.1 U POL-β and 5 nM T4 DNA ligase in buffer containing 20 µCi of [α- 32 P]dATP, 50 µM dNTPs, 50 mM HEPES–KOH (pH 7.6), 30 mM NaCl, 0.1 mg/ml BSA, 2 mM DTT, 2 mM ATP and 3 mM MgCl 2 for 5 and 30 min at 37°C. Lane 1, 30 min in the absence of hTDG and T4 DNA ligase; lane 2, 5 min in the absence of T4 DNA ligase; lane 3, same as 2, but 30 min; lane 4, 30 min in the presence of all proteins. For details see ‘Materials and Methods’ section.
Techniques Used: In Vitro, Incubation
7) Product Images from "Mechanical properties of DNA-like polymers"
Article Title: Mechanical properties of DNA-like polymers
Journal: Nucleic Acids Research
doi: 10.1093/nar/gkt808
Figure Legend Snippet: Example measurement of mechanical properties. ( A ) Cyclization time course for 207-bp DNA-like polymer 5 (pJ1744). DNA ligase-catalyzed cyclization reaction was performed at ∼22°C with 1 nM DNA restriction fragment, T4 DNA ligation buffer (50 mM Tris–HCl, pH 7.5, 10 mM MgCl 2 , 1 mM ATP, 10 mM dithiothreitol) and a final concentration of 100 U/ml T4 DNA ligase. Aliquots (10 µl) were removed at 1–15 min time points, quenched by addition of EDTA to 20 mM and then analyzed by electrophoresis through 5% native polyacrylamide gels in 0.5× TBE buffer (50 mM Tris base, 55 mM boric acid and 1 mM EDTA, pH 8.3). Gel lanes contains Invitrogen 100 bp DNA ladder (M), linear monomer without ligase (0) and increasing 1-min time points of the ligation reaction ( 1–15 ) showing the evolution of linear monomer ( M ), linear dimer ( D ), circular monomer ( C M ) and circular dimer ( C D ). Nearest molecular weight bands are indicated. ( B ) Cyclization kinetics analysis for 207-bp DNA-like polymer 5 (pJ1744). Fitting of data in (A) determines the J -factor, as previously described ( 30 ) (see also Supplementary Data S3 ). ( C ) WLC analysis for DNA-like polymer 5 . Fit of experimental J -factor data using the WLC model. ( D ). Monte Carlo estimation of uncertainty. Fit of simulated J -factor data based on (C) using the WLC model.
Techniques Used: DNA Ligation, Concentration Assay, Electrophoresis, Ligation, Molecular Weight
8) 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
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
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
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:
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
9) 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
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
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
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:
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
10) Product Images from "Profiling the selectivity of DNA ligases in an array format with mass spectrometry"
Article Title: Profiling the selectivity of DNA ligases in an array format with mass spectrometry
Journal: Nucleic Acids Research
doi: 10.1093/nar/gkp827
Figure Legend Snippet: SAMDI was used to assess the relative activity of T4 DNA ligase for substrates having ( A ) deletions and ( B ) insertions of nucleotides at the nick site. In the former cases, spectra are shown for monolayers presenting a matched full length substrate, a single base deletion, a double base deletion and a substrate that does not contain the P1 probe strand. In panel B, spectra are shown for monolayers presenting substrates having an insertion of each of the four nucleotides at the nick site. The star symbol indicates the peak corresponding to the 20-mer DNA strand used for calibrating the mass range. The dotted blue and red lines indicate adenylated and ligated product, respectively.
Techniques Used: Activity Assay
Figure Legend Snippet: An array of substrates was profiled with the T4 DNA ligase and each of four ATP analogs. Experiments were performed and analyzed as described in Figure 3 . Separate experiments were performed using ( A ) ATP, ( B ) ATP-αS, ( C ) ATP-γS and ( D ) AMP-PNP as the cofactor.
Techniques Used:
11) Product Images from "A junction branch point adjacent to a DNA backbone nick directs substrate cleavage by Saccharomyces cerevisiae Mus81-Mms4"
Article Title: A junction branch point adjacent to a DNA backbone nick directs substrate cleavage by Saccharomyces cerevisiae Mus81-Mms4
Journal: Nucleic Acids Research
doi: 10.1093/nar/gkp038
Figure Legend Snippet: Mapping of Mus81-Mms4 incision sites at high protein to substrate ratio. Denaturing urea–PAGE analysis of substrate incision sites on RF-like, nXO12, D-loop (DL) and five 3′-FL-related structures. Assays were performed with 50 nM His10-FLAG-Mus81/GST-Mms4, 50 nM substrate, 30 min at 30°C. Where indicated, reactions were then supplemented with 0.5 mM ATP/3 mM Mg(OAc) 2 and 10 U T4 DNA ligase, with incubation at room temperature for 15 min. All assays were terminated by boiling at 95°C, 2 min, with immediate transfer to ice. ‘L’ represents an oligonucleotide size ladder. The scheme on the right side of the gel illustrates the substrate and cleavage site; the star denotes the position of the 5′ label.
Techniques Used: Polyacrylamide Gel Electrophoresis, Incubation
Figure Legend Snippet: Mus81-Mms4 incises joint molecules at the branch point, adjacent to a phosphodiester backbone discontinuity. Denaturing urea–PAGE analysis of substrate incision sites on RF-like, nXO12, D-loop (DL) and five 3′-FL-related structures. Assays were performed with 10 nM His10-FLAG-Mus81/GST-Mms4, 50 nM substrate, 30 min at 30°C. Where indicated, reactions were then supplemented with 0.5 mM ATP/3 mM Mg(OAc) 2 and 10 U T4 DNA ligase, with incubation at room temperature for 15 min. All assays were terminated by boiling at 95°C, 2 min, with immediate transfer to ice. Nicked duplex DNA without a 5′-phosphate (*) is unligated by T4 DNA ligase, whereas nicked duplex DNA with a 5′ phosphate ( ** ) is ligated by T4 DNA ligase. ‘L’ represents an oligonucleotide size ladder. The scheme on the right side of the gel illustrates the substrate and cleavage site; the star denotes the position of the 5′ label.
Techniques Used: Polyacrylamide Gel Electrophoresis, Incubation
12) 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
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
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
13) Product Images from ""
Article Title:
Journal: The Journal of Biological Chemistry
doi: 10.1074/jbc.M111.284992
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:
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
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 "Blocking of targeted microRNAs from next-generation sequencing libraries"
Article Title: Blocking of targeted microRNAs from next-generation sequencing libraries
Journal: Nucleic Acids Research
doi: 10.1093/nar/gkv724
Figure Legend Snippet: Modification of miRNA sequencing library generation protocol to allow for blocking of targeted species. ( A ) In the standard protocol, a pre-adenylated adaptor is ligated to the 3′ end of a small RNA pool using T4 RNA Ligase 2, truncated. Subsequently, a second adaptor is added to the 5′ end of the miRNA with T4 RNA Ligase 1, followed by reverse transcription and PCR. ( B ) In our modification, a hairpin oligonucleotide with an overhang complementary to the 5′ end of the targeted miRNA is attached via ligation with T4 DNA Ligase to the 5′ end of the miRNA subsequent to the ligation of the adaptor to the 3′ end. This prevents the ligation of the second adaptor to the 5′ end of the miRNA, resulting in a product that does not amplify during PCR. ( C ) Sequencing libraries were generated from human heart total RNA using a titration of a blocking oligonucleotide targeting hsa-miR-16–5p. The fraction of hsa-miR-16–5p present in the blocked library compared to the unblocked library is shown on the y-axis.
Techniques Used: Modification, Sequencing, Blocking Assay, Polymerase Chain Reaction, Ligation, Generated, Titration
15) Product Images from "XRCC4/XLF Interaction Is Variably Required for DNA Repair and Is Not Required for Ligase IV Stimulation"
Article Title: XRCC4/XLF Interaction Is Variably Required for DNA Repair and Is Not Required for Ligase IV Stimulation
Journal: Molecular and Cellular Biology
doi: 10.1128/MCB.01503-14
Figure Legend Snippet: XLF L115A does not interact with XRCC4; thus, it does not bridge DNA in vitro but is fully sufficient to stimulate XRCC4/Lig4. (A, left) Schematic of the DNA bridging assay. (Right) Agarose gel showing recovery of DNA fragments bound to streptavidin beads by ethidium bromide staining. Molecular size markers are indicated (kilobases). (B to D) Ethidium bromide staining of agarose gels showing ligation products obtained from in vitro ligation reactions as described in Materials and Methods. Molecular size markers are indicated (kilobases). (B) T4 DNA ligase is utilized. (C) XRCC4/Lig4 complexes (0.4 μM) are utilized. Four different concentrations of XLF were utilized: 0.25 μM, 0.5 μM, 1 μM, and 2 μM. (D) XRCC4/Lig4 complexes (0.2 μM) are utilized, with wild-type or mutant XRCC4 as indicated and with wild-type or mutant XLF (0.5 μM). (E, top) Immunoblot analyses of lysates from 293 cells transiently transfected with His-tagged wt and mutant forms of XLF probed with antibodies to XRCC4, XLF, or Lig4. (Bottom) Immunoblot analyses of pulldown fractions recovered from Ni-NTA–agarose beads after 3 h of incubation of cell lysates with beads and subsequent washing. The immunoblot was probed with antibodies to XRCC4, XLF, or Lig4.
Techniques Used: In Vitro, Agarose Gel Electrophoresis, Staining, Ligation, Mutagenesis, Transfection, Incubation
16) Product Images from "Highly Efficient One-Step Scarless Protein Tagging by Type IIS Restriction Endonuclease-Mediated Precision Cloning"
Article Title: Highly Efficient One-Step Scarless Protein Tagging by Type IIS Restriction Endonuclease-Mediated Precision Cloning
Journal: Biochemical and biophysical research communications
doi: 10.1016/j.bbrc.2017.05.153
Figure Legend Snippet: Universal and multiple tandem precision tagging strategy ( a ) A template clone contains a TIIS DNA cassette (shaded in blue). Different tags such as GFP (green), Strep-tag II (red), and SNAPf (blue) contain the same two custom sticky ends that belong to SP and gene of interest (indicated by overhangs in black). TIIS restriction enzymes and T4 DNA ligase join them together in a universal manner. ( b ) One sticky end on the right side of GFP is complementary to another one on the left side of Strep-tag II (indicated by green color bar). Similarly, Strep-tag II has a complementary sticky end to the one in SNAPf (indicated by red color bar). TIIS restriction enzyme and T4 ligase can fuse all those tags together and generate a multiple tandem tag (indicated by sequentially arranged GFP, Strep-tag II and SNAPf).
Techniques Used: Strep-tag
17) Product Images from "Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates ▿"
Article Title: Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates ▿
Journal: Journal of Clinical Microbiology
doi: 10.1128/JCM.02340-10
Figure Legend Snippet: Replication capacity of the HBV genome linearized by ApaI and SphI restriction enzymes. The EcoRI dimer of clone 4B was digested with ApaI or SphI, with or without further treatment with T4 DNA ligase before transfection into Huh7 cells. The uncut dimer
Techniques Used: Transfection
Figure Legend Snippet: Functional characterization of two High Fidelity plus PCR clones of the 4B genome. The two clones were transfected directly (lanes 1 and 4) following digestion with BspQI (lanes 2 and 5) or BspQI digestion plus treatment with T4 DNA ligase (lanes 3 and
Techniques Used: Functional Assay, Polymerase Chain Reaction, Clone Assay, Transfection
18) Product Images from "Preparation of Mammalian Expression Vectors Incorporating Site-Specifically Platinated-DNA Lesions"
Article Title: Preparation of Mammalian Expression Vectors Incorporating Site-Specifically Platinated-DNA Lesions
Journal: Bioconjugate chemistry
doi: 10.1021/bc900031a
Figure Legend Snippet: Ligation experiment of gapped pGLuc1temGG (left) and pGLuc2temGTG (right) in presence of different insertion strands with T4 DNA Ligase at 16°C for 12 h; lane 1: gapped pGLuc1temGG alone, lane 2: plasmid + 13-is, lane 3: plasmid + 13-is-Pt, lane
Techniques Used: Ligation, Plasmid Preparation
19) Product Images from "Optimizing the design of protein nanoparticles as carriers for vaccine applications"
Article Title: Optimizing the design of protein nanoparticles as carriers for vaccine applications
Journal: Nanomedicine : nanotechnology, biology, and medicine
doi: 10.1016/j.nano.2015.05.003
Figure Legend Snippet: (a) PDB search for protein structures with inter-helical angles similar to the mode of the nanoparticle followed by superposition of the helices of the different pdb-structures (blue and red) onto the pentamer (green) and trimer (blue) helices of the monomer of the peptide nanoparticle. The residues with angles similar to the angle between the helices of the pentamer and the trimer in the peptide nanoparticle were selected (gray region). ( b) Schematics of the molecular biology strategy for inserting the new linker region: the original construct with pentameric (green) and trimeric (blue) helices is double digested with restriction enzymes ApaI and XhoI. ( c) Linker oligonucleotides selected from panel a are ligated into double digested vector (panel b ) using T4 DNA ligase generating new plasmid which codes for the genetically modified single polypeptide chain.
Techniques Used: Construct, Plasmid Preparation, Genetically Modified
20) Product Images from "Bacillus subtilis LrpC is a sequence-independent DNA-binding and DNA-bending protein which bridges DNA"
Article Title: Bacillus subtilis LrpC is a sequence-independent DNA-binding and DNA-bending protein which bridges DNA
Journal: Nucleic Acids Research
doi:
![DNA circularisation by LrpC. The 182 bp [γ- 32 P]DNA fragment (0.5 nM) was incubated with increasing concentrations of the LrpC protein (30–500 nM, lanes 2–6 and 8–12) in buffer C containing 50 mM NaCl and 1 mM ATP for 15 min at 37°C. In samples 1–6 the DNA was further incubated with ligase buffer and in lanes 7–12 with 1 U of T4 DNA ligase for 30 min at 16°C. The samples were deproteinised and analysed by 4% non-denaturing PAGE in 0.5× TBE, and autoradiographs of the dried gels were subsequently taken.](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC102501/bin/gkd12804.jpg "... and in lanes 7–12 with 1 U of T4 DNA ligase for 30 min at 16°C. The ...")
Figure Legend Snippet: DNA circularisation by LrpC. The 182 bp [γ- 32 P]DNA fragment (0.5 nM) was incubated with increasing concentrations of the LrpC protein (30–500 nM, lanes 2–6 and 8–12) in buffer C containing 50 mM NaCl and 1 mM ATP for 15 min at 37°C. In samples 1–6 the DNA was further incubated with ligase buffer and in lanes 7–12 with 1 U of T4 DNA ligase for 30 min at 16°C. The samples were deproteinised and analysed by 4% non-denaturing PAGE in 0.5× TBE, and autoradiographs of the dried gels were subsequently taken.
Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis
21) Product Images from "Probing transient protein-mediated DNA linkages using nanoconfinement"
Article Title: Probing transient protein-mediated DNA linkages using nanoconfinement
Journal: Biomicrofluidics
doi: 10.1063/1.4882775
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
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:
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:
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:
22) Product Images from "Microfluidic Exponential Rolling Circle Amplification for Sensitive microRNA Detection Directly from Biological Samples"
Article Title: Microfluidic Exponential Rolling Circle Amplification for Sensitive microRNA Detection Directly from Biological Samples
Journal: Sensors and actuators. B, Chemical
doi: 10.1016/j.snb.2018.09.121
Figure Legend Snippet: Optimization of eRCA conditions using the adapter sequence. ( A, B ) Plots of fluorescence signals normalized to the maximum signal obtained under different concentrations of ( A ) the padlock probe and ( B ) T4 DNA ligase. ( C ) Comparison of the fluorescent signals of RCA with and without adding Nb.BbvCI (1 U per 20 μL reaction). ( D, E ) Bar graphs showing the effects of ( D ) reaction time and ( E ) the SYBR Green on the assay response. The 20 μL reaction solution contained 10 −8 M padlock probe and 50 nM let-7a adapter. The ligation reactions were performed at 37 °C for 2 h, and RCA reactions were performed at 30 °C. Error bars represent one standard deviation from three replicates.
Techniques Used: Sequencing, Fluorescence, SYBR Green Assay, Ligation, Standard Deviation
23) Product Images from "Antisense-mediated decrease in DNA ligase III expression results in reduced mitochondrial DNA integrity"
Article Title: Antisense-mediated decrease in DNA ligase III expression results in reduced mitochondrial DNA integrity
Journal: Nucleic Acids Research
doi:
Figure Legend Snippet: T4 DNA ligase treatment of mtDNA from control and AS1 cells. ( A ) Genomic DNA control and AS1 cells electrophoresed under denaturing conditions and hybridized with a mtDNA probe. –, no pretreatment; +, purified DNA treated with T4 DNA ligase prior to denaturation and electrophoresis. ( B ) Scanning densitometry was performed on the data presented above. (Left) DNA from control cell line; (right) DNA from AS1 cell line. Filled circles, pre-treatment with T4 DNA ligase; open circles, no pre-treatment with T4 DNA ligase. The sizes of the fragments were calculated based on the mobility of the band compared to a molecular weight standard.
Techniques Used: Purification, Electrophoresis, Molecular Weight
24) Product Images from "Antisense-mediated decrease in DNA ligase III expression results in reduced mitochondrial DNA integrity"
Article Title: Antisense-mediated decrease in DNA ligase III expression results in reduced mitochondrial DNA integrity
Journal: Nucleic Acids Research
doi:
Figure Legend Snippet: T4 DNA ligase treatment of mtDNA from control and AS1 cells. ( A ) Genomic DNA control and AS1 cells electrophoresed under denaturing conditions and hybridized with a mtDNA probe. –, no pretreatment; +, purified DNA treated with T4 DNA ligase prior to denaturation and electrophoresis. ( B ) Scanning densitometry was performed on the data presented above. (Left) DNA from control cell line; (right) DNA from AS1 cell line. Filled circles, pre-treatment with T4 DNA ligase; open circles, no pre-treatment with T4 DNA ligase. The sizes of the fragments were calculated based on the mobility of the band compared to a molecular weight standard.
Techniques Used: Purification, Electrophoresis, Molecular Weight
25) Product Images from "Ligation with Nucleic Acid Sequence-Based Amplification"
Article Title: Ligation with Nucleic Acid Sequence-Based Amplification
Journal: The Journal of Molecular Diagnostics : JMD
doi: 10.1016/j.jmoldx.2012.01.004
Figure Legend Snippet: A: The LNASBA assay with target or SNP target at a concentration of 0.15 μmol/L. P3 and P5 concentrations were each varied between 0 and 0.35 μmol/L. In the presence of SNP target, T4 DNA ligase does not ligate P3 and P5 together. Only lanes with target show the expected product band (Lanes 4, 6, and 8), highlighted by boxed area . B: Graph showing the effect of increasing P3 and P5 starting concentrations on the 72-nt RNA product concentration. Despite the decreased intensity bands on the pseudogel, there is an increase in product concentration with increased P3 and P5 concentrations.
Techniques Used: Concentration Assay
26) Product Images from "Isolation of Recombinant Adeno-Associated Virus Vector-Cellular DNA Junctions from Mouse Liver"
Article Title: Isolation of Recombinant Adeno-Associated Virus Vector-Cellular DNA Junctions from Mouse Liver
Journal: Journal of Virology
doi:
Figure Legend Snippet: (A) Schematic structure of possible forms of rAAV in tissue (center row), plasmids possibly rescuable by Bam HI digestion and religation (top row), and plasmids possibly rescuable by Bam HI digestion and religation following Pme I digestion and CIP treatment (bottom row). Examples of possible forms in tissue are episomal circular rAAV forms (monomers circularized at the ITRs and aberrant rAAV monomers lacking both Bam HI and Pme I sites), rAAV provirus with three tandem repeats forming a head-to-tail concatemer, and a tail-to-tail junction from either episomal or integrated rAAV forms. Pme I digestion can remove GFP + episomal forms and head-to-tail circular molecules derived from inner repeats of rAAV concatemers. B, Bam HI; P, Pme I site. The straight and zigzag lines represent rAAV vector and mouse genomic DNA sequences, respectively. (B) Construction of a rAAV vector-cellular DNA junction fragment library from mouse liver DNA transduced with AAV-EF1α-GFP.AOSP. High-molecular-weight DNA was isolated from transduced liver. Seven micrograms of liver DNA was digested with Pme I and treated with CIP. DNA (2.25 μg) was directly used to transform E. coli to assess contamination of episomal circular forms of rAAV insensitive to Pme I digestion, and 0.75 μg of DNA was analyzed by gel electrophoresis. Three micrograms of the above-mentioned DNA was digested with Bam HI and self-ligated with T4 DNA ligase. A junction fragment library was made by transforming E. coli with 2.25 μg of the above-mentioned DNA, and the remaining 0.75 μg was electrophoresed to analyze the DNA. Colonies were classified as GFP + (green) and GFP − (white) by UV excitation. The white colonies were subjected to the screening procedure for identification of integrant candidates.
Techniques Used: Derivative Assay, Plasmid Preparation, Transduction, Molecular Weight, Isolation, Nucleic Acid Electrophoresis
27) Product Images from "Small Abundant DNA Binding Proteins from the Thermoacidophilic Archaeon Sulfolobus shibatae Constrain Negative DNA Supercoils"
Article Title: Small Abundant DNA Binding Proteins from the Thermoacidophilic Archaeon Sulfolobus shibatae Constrain Negative DNA Supercoils
Journal: Journal of Bacteriology
doi:
Figure Legend Snippet: Nick closure analysis of the capacity of Ssh7 to constrain DNA supercoils. Plasmid pUC18 containing a single nick per molecule was mixed with Ssh7 at various Ssh7/DNA mass ratios and, after incubation, treated with T4 DNA ligase. The samples were deproteinized and subjected to agarose gel electrophoresis in the presence of 0.5 μg of chloroquine per ml (b) or in the absence of chloroquine (a). Lane A, single-nicked pUC18; lanes B to I, topoisomers of pUC18 ligated at the following Ssh7/DNA mass ratios: 0, 0.22, 0.44, 0.66, 0.88, 1.1, 1.65, and 2.2, respectively; lane J, native pUC18.
Techniques Used: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis
Figure Legend Snippet: Linking number change of circular plasmid DNA covalently closed in the presence of Ssh7. Single-nicked plasmid pUC18 complexed with Ssh7 at various Ssh7/DNA ratios was ligated with T4 DNA ligase. The linking change of the plasmid was measured by resolving topoisomers on agarose gels in the presence of 0.5 to 6 μg of chloroquine per ml or in the absence of chloroquine and band counting.
Techniques Used: Plasmid Preparation
28) Product Images from "The longevity SNP rs2802292 uncovered: HSF1 activates stress-dependent expression of FOXO3 through an intronic enhancer"
Article Title: The longevity SNP rs2802292 uncovered: HSF1 activates stress-dependent expression of FOXO3 through an intronic enhancer
Journal: Nucleic Acids Research
doi: 10.1093/nar/gky331
Figure Legend Snippet: HSF1 mediates the occurrence of a promoter–enhancer interaction at FOXO3 locus involving the 5′UTR and the rs2802292 region. ( A ) Top: physical map of the human FOXO3 gene. The scheme shows the Csp6I restriction enzyme sites flanking the baits (red for the 5′UTR and blue for the rs2802292 region). Bottom: schematic representation of the 3C and ChIP-loop assay. Crosslinked chromatin was digested with Csp6I and immunoprecipitated with anti-HSF1. The immunoprecipitated samples were diluted in a ligation buffer and ligated with the T4 DNA Ligase. After reversing the crosslinks, the ligated DNA was purified and amplified by PCR with various combinations of primers as indicated in (B). ( B ) This strategy allows the amplification of sequences ligated to the bait in circular DNA. The arrows indicate the positions of the primers within the bait sequence. Five different couples of primers were designed to analyze the five possible ligation products. Purified DNA was analyzed by PCR with primers specific for the various possible combinations of chromatin fragments. The values are the results of the densitometric analysis and are expressed as fold induction. HEK-293 cells and primary human fibroblasts (GG, n = 3; TT, n = 3) were collected after induction of oxidative stress (1 h H 2 O 2 , 100 μM). The presented results are representative of three independent experiments. P -values were derived from t -tests: * P ≤ 0.05.
Techniques Used: Chromatin Immunoprecipitation, Immunoprecipitation, Ligation, Purification, Amplification, Polymerase Chain Reaction, Sequencing, Derivative Assay
29) Product Images from "A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL"
Article Title: A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL
Journal: Nucleic Acids Research
doi: 10.1093/nar/gky708
Figure Legend Snippet: RNase H1 processing coupled to POLγ dependent DNA synthesis does not produce ligatable nicks. ( A ) Schematic of the coupled nuclease gap-filling ligation assay performed on a gapped OriL substrate (a). The upstream oligonucleotide was radioactively labelled at the 5′-end. The possible products are illustrated (b–d). (–) Coupled nuclease gap-filling ligation assay as shown in A. POLγ filled the gap (lane 2, marked b) and had limited strand displacement activity (SD). Note though that POLγ completely displaces the downstream oligonucleotide in a small fraction of templates (80 nt band, lanes 3–6,), RNase H1 cleaved the RNA in the substrate (lane 3), enabling further gap-filling (marked c). Only very low levels of ligated products were formed in the presence of 80–320 fmol DNA ligase III (lanes 4–6). A prominent 80 nt ligated product was formed with T4 DNA ligase (lane 7, marked d). The letters a-d correspond to the illustrations in panel A. ( C ) Ligation assay on a nicked substrate containing RNA tracts of varying length downstream of the nick in the presence of 80–320 fmol DNA ligase III. DNA ligase III discriminates against nicked substrates that contain increasing stretches of ribonucleotides. Two, but not five or more ribonucleotides, can be ligated. ( D ) As in C, except performed with T4 ligase (1–8 U). T4 ligase can ligate five but not 10 ribonucleotides. ( E ) T4 ligase-mediated ligation is abolished in the presence of the mutant RNase H1 proteins. The letters a-d correspond to the illustrations in panel A.
Techniques Used: DNA Synthesis, Ligation, Activity Assay, Mutagenesis
30) Product Images from "A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL"
Article Title: A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL
Journal: Nucleic Acids Research
doi: 10.1093/nar/gky708
Figure Legend Snippet: RNase H1 processing coupled to POLγ dependent DNA synthesis does not produce ligatable nicks. ( A ) Schematic of the coupled nuclease gap-filling ligation assay performed on a gapped OriL substrate (a). The upstream oligonucleotide was radioactively labelled at the 5′-end. The possible products are illustrated (b–d). (–) Coupled nuclease gap-filling ligation assay as shown in A. POLγ filled the gap (lane 2, marked b) and had limited strand displacement activity (SD). Note though that POLγ completely displaces the downstream oligonucleotide in a small fraction of templates (80 nt band, lanes 3–6,), RNase H1 cleaved the RNA in the substrate (lane 3), enabling further gap-filling (marked c). Only very low levels of ligated products were formed in the presence of 80–320 fmol DNA ligase III (lanes 4–6). A prominent 80 nt ligated product was formed with T4 DNA ligase (lane 7, marked d). The letters a-d correspond to the illustrations in panel A. ( C ) Ligation assay on a nicked substrate containing RNA tracts of varying length downstream of the nick in the presence of 80–320 fmol DNA ligase III. DNA ligase III discriminates against nicked substrates that contain increasing stretches of ribonucleotides. Two, but not five or more ribonucleotides, can be ligated. ( D ) As in C, except performed with T4 ligase (1–8 U). T4 ligase can ligate five but not 10 ribonucleotides. ( E ) T4 ligase-mediated ligation is abolished in the presence of the mutant RNase H1 proteins. The letters a-d correspond to the illustrations in panel A.
Techniques Used: DNA Synthesis, Ligation, Activity Assay, Mutagenesis
31) Product Images from "Assessing Protein Dynamics on Low-Complexity Single-Stranded DNA Curtains"
Article Title: Assessing Protein Dynamics on Low-Complexity Single-Stranded DNA Curtains
Journal: Langmuir : the ACS journal of surfaces and colloids
doi: 10.1021/acs.langmuir.8b01812
Figure Legend Snippet: Assembly of low-complexity ssDNA curtains. (A) A phosphorylated template (black) and a biotinylated primer (green) are annealed and treated with T4 DNA ligase to make minicircles. Low-complexity ssDNA composed solely of thymidine and cytidine is synthesized via rolling circle replication by phi29 DNAP. (B) Low-complexity ssDNA curtains with fluorescent end labeling. The 3′ end of the ssDNA was labeled with a fluorescent antibody. (C) RPA-GFP (green)-coated ssDNA with fluorescent end labeling (magenta). (D) Kymograph of a representative ssDNA in panel (C) with buffer flow on and off, indicating that the ssDNA is anchored to the surface via the 5′-biotin tether.
Techniques Used: Synthesized, End Labeling, Labeling, Recombinase Polymerase Amplification, Flow Cytometry
32) Product Images from "Gain of function mutant p53 proteins cooperate with E2F4 to transcriptionally downregulate RAD17 and BRCA1 gene expression"
Article Title: Gain of function mutant p53 proteins cooperate with E2F4 to transcriptionally downregulate RAD17 and BRCA1 gene expression
Journal: Oncotarget
doi:
Figure Legend Snippet: BRCA1 expression counteracts mutant p53 GOF activity on DNA repair assay (A) Comparison of ligation products of 5′-cohesive-ended linear DNA in the presence of T4 DNA ligase alone (lane 3) or following pre-incubation with whole protein extracts derived from H1299 cells transfected with mutp53R175H and BRCA1 expressing vectors in separate reactions (lanes 5 and 7, respectively) or in co-trasfection conditions (lane 6). (B) Whole protein extracts (40 μg) used in the T4 DNA ligase assay previously described were subjected to Western blot analysis and probed with the indicated antibodies. (C-E) SKBr3 cells were transiently transfected with ApaI-linearized pSI-CHECK2 vector (c) and with either siRNA oligos indicated in the figures (d) and (e). After 48 h from the transfection the cells were harvested and the functional changes in NHEJ were assessed measuring the Firefly Luciferase activity. Luciferase activity was expressed as (Firefly/protein amount) × (1/Renilla). Columns , means from two independent assays each of them was done in triplicate; bars , SD. P -values were calculated with two tailed t-test. Statistically significant results were with p -value
Techniques Used: Expressing, Mutagenesis, Activity Assay, Ligation, Incubation, Derivative Assay, Transfection, Western Blot, Plasmid Preparation, Functional Assay, Non-Homologous End Joining, Luciferase, Two Tailed Test
33) Product Images from "7,8-dihydro-8-oxoadenine, a highly mutagenic adduct, is repaired by Escherichia coli and human mismatch-specific uracil/thymine-DNA glycosylases"
Article Title: 7,8-dihydro-8-oxoadenine, a highly mutagenic adduct, is repaired by Escherichia coli and human mismatch-specific uracil/thymine-DNA glycosylases
Journal: Nucleic Acids Research
doi: 10.1093/nar/gks1149
Figure Legend Snippet: In vitro reconstitution of the BER pathway using 8oxoA•T duplex DNA substrate. 5 nM 40 mer 8oxoA•T duplex was incubated in the presence of 20 nM hTDG, 5 nM APE1, 2 nM FEN1, 0.1 U POL-β and 5 nM T4 DNA ligase in buffer containing 20 µCi of [α- 32 P]dATP, 50 µM dNTPs, 50 mM HEPES–KOH (pH 7.6), 30 mM NaCl, 0.1 mg/ml BSA, 2 mM DTT, 2 mM ATP and 3 mM MgCl 2 for 5 and 30 min at 37°C. Lane 1, 30 min in the absence of hTDG and T4 DNA ligase; lane 2, 5 min in the absence of T4 DNA ligase; lane 3, same as 2, but 30 min; lane 4, 30 min in the presence of all proteins. For details see ‘Materials and Methods’ section.
Techniques Used: In Vitro, Incubation
34) Product Images from "Rationally designed coiled-coil DNA looping peptides control DNA topology"
Article Title: Rationally designed coiled-coil DNA looping peptides control DNA topology
Journal: Nucleic Acids Research
doi: 10.1093/nar/gkt553
Figure Legend Snippet: Topological effects of protein-induced looping, for ten 862 bp cyclized DNA fragments with CREB and Inv-2 sites separated by 435–458 bp. DNA (0.3 nM) was cyclized by T4 DNA ligase in the presence of LZD73 or LZD87, treated with BAL31 nuclease and analyzed as in Figure 3 . Cyclization of DNA alone is shown for the 448 and 455 constructs on the left; all the other constructs give the same results. The distribution of topoisomers in the presence of LZD peptides, including new positive and negative supercoils, is a periodic function of binding site separation. ( B ) Schematic of the variation of inner and outer loop lengths at a constant 862 bp total length. ( C ) Looping introduces a writhe crossover, which can be of either sign because the binding sites are palindromic. The peptide can also introduce a local ΔTw. ( D ) A ΔTw in the inner lobe is required to bring binding sites into alignment to form a protein-mediated loop, and a ΔTw in the outer loop is required for ring closure. Both twist changes and any writhe in the lobes contribute to the total observed ΔLk.
Techniques Used: Construct, Binding Assay, Introduce
Figure Legend Snippet: Ligation reactions of DNA fragments with CREB and Inv-2 sites separated by variable DNA lengths demonstrate looping through the appearance of new topoisomers on ring closure. DNA fragments were synthesized by PCR from plasmids with CREB and Inv-2-binding sites separated by 153–448 bp. The CREB-XhoI spacing was 203 bp and Inv-2-XhoI was 211 bp except for the Vx(448)212 construct at the far right. ( A ) The LZD peptide could affect the distribution of ligation products in several ways. LZD can enhance bimolecular reactions (i and v), or it can form a loop that alters the cyclization probability and/or the topology of the ligation products through ΔTw and ΔWr (iii and iv), or it may have no effect on cyclization (ii). ( B ) Variable length DNA fragments with XhoI overhangs were treated with T4 DNA ligase. Deproteinized samples were analyzed by native PAGE on a 6% 75:1 gel containing 7.5 μg/ml chloroquine to resolve topoisomers. Each set of six lanes shows starting DNA, ligated DNA, ligation in the presence of the control GCN4 peptide, ligation with LZD73, ligation with LZD87 and ligation with LZD87 followed by BAL31 digestion to identify DNA minicircles. The calculated Lk for cyclized products assumes a DNA helical repeat of 10.55 bp/turn. New positive and negative topoisomers diagnostic for looping are seen only for molecules with ≥310-bp site spacing (inner loops) and > 212-bp outer loops. Slight enhancement of bimolecular ligation is also seen, especially for molecules that do not loop.
Techniques Used: Ligation, Synthesized, Polymerase Chain Reaction, Binding Assay, Construct, Clear Native PAGE, DNA Ligation, Diagnostic Assay
35) Product Images from "Validation and Genotyping of Multiple Human Polymorphic Inversions Mediated by Inverted Repeats Reveals a High Degree of Recurrence"
Article Title: Validation and Genotyping of Multiple Human Polymorphic Inversions Mediated by Inverted Repeats Reveals a High Degree of Recurrence
Journal: PLoS Genetics
doi: 10.1371/journal.pgen.1004208
Figure Legend Snippet: Multiplex iPCR results of two validated inversions in nine human samples. A . HsInv0403 ABD and ACD iPCRs. Band sizes are: AB, 364 bp; BD, 239 bp; AC, 350 bp; CD, 225 bp; and AD, 265 bp. B . HsInv0209 ABC and BCD iPCRs. Band sizes are AB, 435 bp; AC, 243 bp; BD, 543 bp; CD, 351 bp; and BC, 470 bp. For both panels the genomic DNA samples are: 1, negative control; 2, NA12156; 3, NA12878; 4, NA15510; 5, NA18507; 6, NA18517; 7, NA18555; 8, NA18956; 9, NA19129; 10, NA19240; 11, DNA without restriction enzyme; 12, DNA without T4 DNA ligase; and L, 100 bp DNA Ladder (Invitrogen).
Techniques Used: Multiplex Assay, Negative Control
36) Product Images from "Aberrant repair initiated by mismatch-specific thymine-DNA glycosylases provides a mechanism for the mutational bias observed in CpG islands"
Article Title: Aberrant repair initiated by mismatch-specific thymine-DNA glycosylases provides a mechanism for the mutational bias observed in CpG islands
Journal: Nucleic Acids Research
doi: 10.1093/nar/gku246
![In vitro reconstitution of the aberrant BER pathway using TDG and Hx•T duplex. 5 nM 5′-[ 32 P]-labeled oligonucleotide duplex was incubated with 300-nM TDG, 5 nM APE1, 0.1 unit Polβ, 2 units T4 DNA Ligase and 50 μM of dNTPs for 30 min at 37°C. BstUI digestion was carried out at 60°C for 40 min. Lane 1: T•Hx duplex in which T-containing strand is 5′-[ 32 P]-labeled incubated with TDG and then treated by 0.1 M NaOH to cleave at AP sites; lanes 2–8: Hx•T and Hx•C duplexes in which Hx-containing strand is 5′-[ 32 P]-labeled; lanes 9 and 10: G•T duplex in which G-containing strand is 5′-[ 32 P]-labeled. The reaction products were analyzed as described in the Materials and Methods section. Arrows indicate TDG- and BstUI-catalyzed cleavage products.](https://storage.googleapis.com/bioz_article_images/PMC4041421/gku246fig5.jpg "... 5 nM APE1, 0.1 unit Polβ, 2 units T4 DNA Ligase and 50 μM of dNTPs for ...")
Figure Legend Snippet: In vitro reconstitution of the aberrant BER pathway using TDG and Hx•T duplex. 5 nM 5′-[ 32 P]-labeled oligonucleotide duplex was incubated with 300-nM TDG, 5 nM APE1, 0.1 unit Polβ, 2 units T4 DNA Ligase and 50 μM of dNTPs for 30 min at 37°C. BstUI digestion was carried out at 60°C for 40 min. Lane 1: T•Hx duplex in which T-containing strand is 5′-[ 32 P]-labeled incubated with TDG and then treated by 0.1 M NaOH to cleave at AP sites; lanes 2–8: Hx•T and Hx•C duplexes in which Hx-containing strand is 5′-[ 32 P]-labeled; lanes 9 and 10: G•T duplex in which G-containing strand is 5′-[ 32 P]-labeled. The reaction products were analyzed as described in the Materials and Methods section. Arrows indicate TDG- and BstUI-catalyzed cleavage products.
Techniques Used: In Vitro, Labeling, Incubation
37) 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
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
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
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:
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
38) 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
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
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
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:
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
39) 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
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
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
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:
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
40) 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
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
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
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:
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
41) 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
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
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
42) 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
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
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
43) 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
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
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
44) 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
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
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
45) Product Images from "Staphylococcus aureus Host Cell Invasion and Virulence in Sepsis Is Facilitated by the Multiple Repeats within FnBPA"
Article Title: Staphylococcus aureus Host Cell Invasion and Virulence in Sepsis Is Facilitated by the Multiple Repeats within FnBPA
Journal: PLoS Pathogens
doi: 10.1371/journal.ppat.1000964
Figure Legend Snippet: Construction of FnBPA variants containing various repeats. A. Diagrammatic representation of FnBPA, illustrating the relative positions of the N-terminal signal sequence (SS), N-terminal sub-domains (N1, N2 and N3) forming the A domain, 11 Fn-binding repeats (numbered 1–11; those with high affinity for Fn are indicated with asterices), proline-rich region (PRR) and the sortase A recognition sequence LPXTG. The FnBPA variants described in this report differ solely in the composition of the Fn-binding repeat region. Thus FnBPR0 refers to FnBPA without any repeats, FnBPR1 contains only repeat 1 whilst FnBPR1-11 encodes the entire fnb A gene etc. The old organizational scheme is shown below the new one. B. Construction strategy used to produce fnb A variants containing various combinations of repeats. PCR primers with 5′ phosphate were employed to amplify the entire pFNBA4 plasmid, minus the repeats to be excluded. The blunt ended phosphorylated DNA could then be self-ligated using T4 DNA ligase forming a plasmid containing the modified fnb A gene. C. Diagrammatic representation of the composition of the Fn-binding domains of FnBPA variants used in this study. Each square represents a single repeat. Filled squares and open squares represent high- and low-affinity repeats respectively. D. Western immunoblots of surface proteins from S. aureus expressing various FnBPA constructs. FnBPA variants were detected using an antibody that recognizes the N-terminal domain that is common to all the constructs used in this study. Blots are labelled according to the repeats present in each of the FnBPA variants expressed.
Techniques Used: Sequencing, Binding Assay, Polymerase Chain Reaction, Plasmid Preparation, Modification, Western Blot, Expressing, Construct
46) Product Images from "An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution"
Article Title: An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution
Journal: Biological Procedures Online
doi: 10.1251/bpo33
Figure Legend Snippet: Gel-electrophoresis of ligation products. The ligation products were electrophoresed on 8M Urea 10% PAGE at 65 ºC and were visualized with fluorescence of FITC and then visualized after staining by SYBR Green II using an imager. Lane CY: control, Linker-Y. Lane CS: control, mRNA without ligation. Lane CR: control, Linker-S. Lane Y: Y-ligation with T4 RNA ligase. Lane R: splint ligation with T4 RNA ligase. Lane D: splint ligation with T4 DNA ligase.
Techniques Used: Nucleic Acid Electrophoresis, Ligation, Polyacrylamide Gel Electrophoresis, Fluorescence, Staining, SYBR Green Assay
47) Product Images from ""
Article Title:
Journal: The Journal of Biological Chemistry
doi: 10.1074/jbc.M111.284992
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:
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
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:
48) Product Images from ""
Article Title:
Journal: The Journal of Biological Chemistry
doi: 10.1074/jbc.M111.284992
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:
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
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:
49) Product Images from ""
Article Title:
Journal: The Journal of Biological Chemistry
doi: 10.1074/jbc.M111.284992
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:
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
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:
50) Product Images from ""
Article Title:
Journal: The Journal of Biological Chemistry
doi: 10.1074/jbc.M111.284992
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:
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
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:
51) Product Images from ""
Article Title:
Journal: The Journal of Biological Chemistry
doi: 10.1074/jbc.M111.284992
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:
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
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:
52) Product Images from "Temperature dependence of DNA persistence length"
Article Title: Temperature dependence of DNA persistence length
Journal: Nucleic Acids Research
doi: 10.1093/nar/gkq932
Figure Legend Snippet: Temperature dependence of . The distributions of the topoisomers were obtained by applying T4 DNA ligase (open circle) or Taq DNA ligase (open triangle) to nicked samples of pUC19 plasmid.
Techniques Used: Plasmid Preparation
Figure Legend Snippet: Dependence of the measured C/D on the concentration of T4 DNA ligase. The shown data correspond to the fragments with EcoRI (5°C, filled circle) and HindIII (42°C, filled triangle) sticky ends. The data show that the ratio does not change if the ligase concentration is below 100 U/ml.
Techniques Used: Concentration Assay
53) Product Images from "Temperature dependence of DNA persistence length"
Article Title: Temperature dependence of DNA persistence length
Journal: Nucleic Acids Research
doi: 10.1093/nar/gkq932
Figure Legend Snippet: Temperature dependence of . The distributions of the topoisomers were obtained by applying T4 DNA ligase (open circle) or Taq DNA ligase (open triangle) to nicked samples of pUC19 plasmid.
Techniques Used: Plasmid Preparation
Figure Legend Snippet: Dependence of the measured C/D on the concentration of T4 DNA ligase. The shown data correspond to the fragments with EcoRI (5°C, filled circle) and HindIII (42°C, filled triangle) sticky ends. The data show that the ratio does not change if the ligase concentration is below 100 U/ml.
Techniques Used: Concentration Assay
54) Product Images from "Blocking of targeted microRNAs from next-generation sequencing libraries"
Article Title: Blocking of targeted microRNAs from next-generation sequencing libraries
Journal: Nucleic Acids Research
doi: 10.1093/nar/gkv724
Figure Legend Snippet: Modification of miRNA sequencing library generation protocol to allow for blocking of targeted species. ( A ) In the standard protocol, a pre-adenylated adaptor is ligated to the 3′ end of a small RNA pool using T4 RNA Ligase 2, truncated. Subsequently, a second adaptor is added to the 5′ end of the miRNA with T4 RNA Ligase 1, followed by reverse transcription and PCR. ( B ) In our modification, a hairpin oligonucleotide with an overhang complementary to the 5′ end of the targeted miRNA is attached via ligation with T4 DNA Ligase to the 5′ end of the miRNA subsequent to the ligation of the adaptor to the 3′ end. This prevents the ligation of the second adaptor to the 5′ end of the miRNA, resulting in a product that does not amplify during PCR. ( C ) Sequencing libraries were generated from human heart total RNA using a titration of a blocking oligonucleotide targeting hsa-miR-16–5p. The fraction of hsa-miR-16–5p present in the blocked library compared to the unblocked library is shown on the y-axis.
Techniques Used: Modification, Sequencing, Blocking Assay, Polymerase Chain Reaction, Ligation, Generated, Titration
55) Product Images from "YY1 Is a Structural Regulator of Enhancer-Promoter Loops"
Article Title: YY1 Is a Structural Regulator of Enhancer-Promoter Loops
Journal: Cell
doi: 10.1016/j.cell.2017.11.008
Figure Legend Snippet: YY1 Can Enhance DNA Interactions In Vitro (A and D) Models depicting the in vitro DNA circularization assays used to detect the ability of YY1 to enhance DNA looping interactions with no motif control (A) or competitor DNA control (D). (B and E) Results of the in vitro DNA circularization assay visualized by gel electrophoresis with no motif control (B) or competitor DNA control (E). The dominant lower band reflects the starting linear DNA template, while the upper band corresponds to the circularized DNA ligation product. (C and F) Quantifications of DNA template circularization as a function of incubation time with T4 DNA ligase for no motif control (C) or competitor DNA control (F). Values correspond to the percent of DNA template that is circularized and represents the mean and SD of four experiments. .
Techniques Used: In Vitro, Nucleic Acid Electrophoresis, DNA Ligation, Incubation
56) Product Images from "Enhancement of DNA flexibility in vitro and in vivo by HMGB box A proteins carrying box B residues"
Article Title: Enhancement of DNA flexibility in vitro and in vivo by HMGB box A proteins carrying box B residues
Journal: Biochemistry
doi: 10.1021/bi802269f
Figure Legend Snippet: In vitro assay of DNA flexibility enhancement by HMGB proteins and chimeras. A. Example data from T4 DNA ligase cyclization assay for 200-bp DNA probe in the absence (—) and presence of 40 nM HMGB constructs 16 and 5 (see ). B. Graphical
Techniques Used: In Vitro, Construct
57) Product Images from "Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates ▿"
Article Title: Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates ▿
Journal: Journal of Clinical Microbiology
doi: 10.1128/JCM.02340-10
Figure Legend Snippet: Replication capacity of the HBV genome linearized by ApaI and SphI restriction enzymes. The EcoRI dimer of clone 4B was digested with ApaI or SphI, with or without further treatment with T4 DNA ligase before transfection into Huh7 cells. The uncut dimer
Techniques Used: Transfection
Figure Legend Snippet: Functional characterization of two High Fidelity plus PCR clones of the 4B genome. The two clones were transfected directly (lanes 1 and 4) following digestion with BspQI (lanes 2 and 5) or BspQI digestion plus treatment with T4 DNA ligase (lanes 3 and
Techniques Used: Functional Assay, Polymerase Chain Reaction, Clone Assay, Transfection
58) Product Images from "Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates ▿"
Article Title: Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates ▿
Journal: Journal of Clinical Microbiology
doi: 10.1128/JCM.02340-10
Figure Legend Snippet: Replication capacity of the HBV genome linearized by ApaI and SphI restriction enzymes. The EcoRI dimer of clone 4B was digested with ApaI or SphI, with or without further treatment with T4 DNA ligase before transfection into Huh7 cells. The uncut dimer
Techniques Used: Transfection
Figure Legend Snippet: Functional characterization of two High Fidelity plus PCR clones of the 4B genome. The two clones were transfected directly (lanes 1 and 4) following digestion with BspQI (lanes 2 and 5) or BspQI digestion plus treatment with T4 DNA ligase (lanes 3 and
Techniques Used: Functional Assay, Polymerase Chain Reaction, Clone Assay, Transfection
59) Product Images from "Preparation of Mammalian Expression Vectors Incorporating Site-Specifically Platinated-DNA Lesions"
Article Title: Preparation of Mammalian Expression Vectors Incorporating Site-Specifically Platinated-DNA Lesions
Journal: Bioconjugate chemistry
doi: 10.1021/bc900031a
Figure Legend Snippet: Ligation experiment of gapped pGLuc1temGG (left) and pGLuc2temGTG (right) in presence of different insertion strands with T4 DNA Ligase at 16°C for 12 h; lane 1: gapped pGLuc1temGG alone, lane 2: plasmid + 13-is, lane 3: plasmid + 13-is-Pt, lane
Techniques Used: Ligation, Plasmid Preparation
60) Product Images from "Probing transient protein-mediated DNA linkages using nanoconfinement"
Article Title: Probing transient protein-mediated DNA linkages using nanoconfinement
Journal: Biomicrofluidics
doi: 10.1063/1.4882775
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
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:
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:
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:
61) Product Images from "Probing transient protein-mediated DNA linkages using nanoconfinement"
Article Title: Probing transient protein-mediated DNA linkages using nanoconfinement
Journal: Biomicrofluidics
doi: 10.1063/1.4882775
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
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:
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:
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:
62) Product Images from "Probing transient protein-mediated DNA linkages using nanoconfinement"
Article Title: Probing transient protein-mediated DNA linkages using nanoconfinement
Journal: Biomicrofluidics
doi: 10.1063/1.4882775
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
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:
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:
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:
63) Product Images from "The unstructured linker arms of MutL enable GATC site incision beyond roadblocks during initiation of DNA mismatch repair"
Article Title: The unstructured linker arms of MutL enable GATC site incision beyond roadblocks during initiation of DNA mismatch repair
Journal: Nucleic Acids Research
doi: 10.1093/nar/gkz834
Figure Legend Snippet: Single molecule DNA nanomanipulation assay for MMR strand incision. ( A ) The heteroduplex Rb-pREP4 DNA substrate is tethered and supercoiled in the magnetic trap in the presence of MMR components. Incision of DNA is observed as an abrupt increase in DNA extension. Drawing is not to scale. ( B ) Time-trace showing repeated cycles of supercoiling and subsequent incision of the substrate by 2.5 nM MutS, 10 nM MutL, 10 nM MutH. In between cycles incised DNA is religated by T4 DNA ligase which is present in the reaction. Blue points show bead position sampled at 31 Hz, red points show raw data with ∼1 s averaging. Green line represents the stepwise increase in supercoiling imposed on the DNA via the magnetic trap. Red arrows indicate incision events. T wait represents the time elapsed before a supercoiled DNA is incised. ( C ) Distribution of T wait is well-described by a single-exponential fit (red line) with mean
Techniques Used:
64) Product Images from "Antisense-mediated decrease in DNA ligase III expression results in reduced mitochondrial DNA integrity"
Article Title: Antisense-mediated decrease in DNA ligase III expression results in reduced mitochondrial DNA integrity
Journal: Nucleic Acids Research
doi:
Figure Legend Snippet: T4 DNA ligase treatment of mtDNA from control and AS1 cells. ( A ) Genomic DNA control and AS1 cells electrophoresed under denaturing conditions and hybridized with a mtDNA probe. –, no pretreatment; +, purified DNA treated with T4 DNA ligase prior to denaturation and electrophoresis. ( B ) Scanning densitometry was performed on the data presented above. (Left) DNA from control cell line; (right) DNA from AS1 cell line. Filled circles, pre-treatment with T4 DNA ligase; open circles, no pre-treatment with T4 DNA ligase. The sizes of the fragments were calculated based on the mobility of the band compared to a molecular weight standard.
Techniques Used: Purification, Electrophoresis, Molecular Weight
65) Product Images from "Inhibitor of caspase-activated DNase expression enhances caspase-activated DNase expression and inhibits oxidative stress-induced chromosome breaks at the mixed lineage leukaemia gene in nasopharyngeal carcinoma cells"
Article Title: Inhibitor of caspase-activated DNase expression enhances caspase-activated DNase expression and inhibits oxidative stress-induced chromosome breaks at the mixed lineage leukaemia gene in nasopharyngeal carcinoma cells
Journal: Cancer Cell International
doi: 10.1186/s12935-015-0205-1
Figure Legend Snippet: Flow chart showing DNA modification and IPCR. The arrow heads indicate the forward and reverse primers that were designed in opposite direction. Bam H I digestion yielded a mixture of intact chromosome and cleaved chromosome. Klenow fill-in produced blunt ended chromosome fragments which were then cyclilsed by T4 DNA ligase. The intact chromosome will become a large circle while the cleaved chromosome will become a smaller circle. Upon cyclisation, the primers are now in correct orientation for amplification. Msc I digestion cleaved both circles outside the amplification region, thus merely linearise the molecule. Amplification from intact MLL gene will produce longer PCR products while amplification from cleaved MLL gene will yield shorter PCR products
Techniques Used: Flow Cytometry, Modification, Produced, Amplification, Polymerase Chain Reaction
66) Product Images from "Quantitative disclosure of DNA knot chirality by high-resolution 2D-gel electrophoresis"
Article Title: Quantitative disclosure of DNA knot chirality by high-resolution 2D-gel electrophoresis
Journal: Nucleic Acids Research
doi: 10.1093/nar/gkz015
Figure Legend Snippet: Test of the electrophoresis procedure that discerns DNA knot chirality. ( A ) A linear 4.4-kb DNA fragment was circularized in free solution with T4 DNA ligase to produce a small fraction of molecules containing a trefoil knot. Negative supercoils were subsequently introduced by incubating the circularized DNA with topoisomerase I in presence of 250 μg/ml chloroquine. ( B ) The gel-blot shows the DNA products after high resolution 2D-gel electrophoresis carried out in 0.9% agarose gel (40 × 23 cm) in TBE. The first gel dimension (top to bottom) was run at 80 V for 70 h in TBE (89 mM Tris-borate, pH 8.3, 2 mM EDTA). The second gel dimension (left to right) was run at 120 V for 10 h in TBE containing 0.65 μg/ml of chloroquine. Lk, linking number topoisomers. N, nicked unknotted circles. L, linear DNA. The enlarged gel section shows the signal of Lk topoisomers of unknotted molecules (Kn# 0) and of molecules containing either a positive- or negative-noded trefoil knot (Kn# 3). ( C ) Probability of the two chiral forms of the trefoil knot.
Techniques Used: Electrophoresis, Western Blot, Two-Dimensional Gel Electrophoresis, Agarose Gel Electrophoresis
67) Product Images from "Iron mediates catalysis of nucleic acid processing enzymes: support for Fe(II) as a cofactor before the great oxidation event"
Article Title: Iron mediates catalysis of nucleic acid processing enzymes: support for Fe(II) as a cofactor before the great oxidation event
Journal: Nucleic Acids Research
doi: 10.1093/nar/gkx171
Figure Legend Snippet: Fe 2+ can replace Mg 2+ as a cofactor for T4 DNA ligase. Ligation products of sliding-half complementary oligonucleotides are observed with both Mg 2+ and Fe 2+ . No product is observed in the divalent-minus negative control. See ‘Materials and Methods’ section for oligonucleotide sequences.
Techniques Used: Ligation, Negative Control
68) Product Images from "The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans"
Article Title: The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans
Journal: PLoS Biology
doi: 10.1371/journal.pbio.2001164
Figure Legend Snippet: Mlh1-Mlh3 can make DSBs on large DNA substrates. (A) Mlh1-Mlh3 makes exclusively linear product from a closed circular substrate approximately 12 kb in size. Experiments were performed identical to the manner described in Fig 2C using the indicated sized plasmids. Red asterisk indicates location where nicked 12 kb plasmid migrates. (B) 15 μM total nucleotide 12 kb circular substrate was incubated with 300 nM Mlh1-Mlh3 for the indicated period of time. Plasmid linearized with Hin dIII was used as a marker for linear product in the lane 8 of the left panel. 12 kb plasmid treated with DNaseI is used as a marker for closed circular, linear, and nicked species in lane 1. Lane 9 is a negative control reaction in which Mlh1-Mlh3 was omitted to indicate migration of closed circular substrate. The plot indicates quantification of the representative gel shown. (C) Experiment is identical to that conducted in B, except 15 μM total nucleotide 2.7 kb circular substrate was used. The plot indicates quantification of the representative gel shown. (D) Native gel analysis of material in Fig 2E lanes 7–11. (E). DSBs made by Mlh1-Mlh3 can be religated. 12 kb linear product from Mlh1-Mlh3 endonuclease assay (“Mlh1-Mlh3 linear pdt”) was gel isolated and incubated with T4 polymerase where indicated with a + (lanes 12–13) followed by T4 DNA ligase where indicated with a + (lanes 11, 13). As controls, 12 kb closed circular plasmid was linearized with either Sca I or Hin dIII and religated (lanes 4–7) or linearized with Hin dIII and blunted with T4 polymerase followed by a religation step (lanes 8–9). Gel-isolated 12 kb closed circular DNA and Sca I-linearized DNA were ran in lanes 2–3 as migration markers.
Techniques Used: Plasmid Preparation, Incubation, Marker, Negative Control, Migration, Isolation
69) 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
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
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
70) Product Images from "A phosphate-targeted dinuclear Cu(II) complex combining major groove binding and oxidative DNA cleavage"
Article Title: A phosphate-targeted dinuclear Cu(II) complex combining major groove binding and oxidative DNA cleavage
Journal: Nucleic Acids Research
doi: 10.1093/nar/gky806
Figure Legend Snippet: ( A ) BDNPP hydrolytic cleavage mechanism in the presence of Cu 2 TPNap; ( B ) Lineweaver–Burk plot; ( C ) rate-pH profile for the cleavage of BDNPP in the presence of Cu 2 TPNap at 40°C; ( D ) DNA cleavage reactions by Cu 2 TPNap on pUC19 plasmid DNA over 1 h at 37°C in the absence of added reductant; and ( E ) T4 DNA ligase experiments with Cu 2 TPNap and restriction enzymes EcoRI and Nt.BspQI.
Techniques Used: Plasmid Preparation
71) Product Images from "Padlock oligonucleotides for duplex DNA based on sequence-specific triple helix formation"
Article Title: Padlock oligonucleotides for duplex DNA based on sequence-specific triple helix formation
Journal: Proceedings of the National Academy of Sciences of the United States of America
doi:
Figure Legend Snippet: Schematic diagram of the locking technique described in this paper. The central part of a linear oligonucleotide (yellow) binds to the major groove of a specific sequence within a plasmid (red and blue) to form a triple-helical complex. Then, its 5′ and 3′ ends hybridize adjacent to each other to a template oligonucleotide (green) and are joined together by T4 DNA ligase (white) to form a circular oligonucleotide catenated to the plasmid DNA.
Techniques Used: Sequencing, Plasmid Preparation
72) Product Images from "A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL"
Article Title: A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL
Journal: Nucleic Acids Research
doi: 10.1093/nar/gky708
Figure Legend Snippet: RNase H1 processing coupled to POLγ dependent DNA synthesis does not produce ligatable nicks. ( A ) Schematic of the coupled nuclease gap-filling ligation assay performed on a gapped OriL substrate (a). The upstream oligonucleotide was radioactively labelled at the 5′-end. The possible products are illustrated (b–d). (–) Coupled nuclease gap-filling ligation assay as shown in A. POLγ filled the gap (lane 2, marked b) and had limited strand displacement activity (SD). Note though that POLγ completely displaces the downstream oligonucleotide in a small fraction of templates (80 nt band, lanes 3–6,), RNase H1 cleaved the RNA in the substrate (lane 3), enabling further gap-filling (marked c). Only very low levels of ligated products were formed in the presence of 80–320 fmol DNA ligase III (lanes 4–6). A prominent 80 nt ligated product was formed with T4 DNA ligase (lane 7, marked d). The letters a-d correspond to the illustrations in panel A. ( C ) Ligation assay on a nicked substrate containing RNA tracts of varying length downstream of the nick in the presence of 80–320 fmol DNA ligase III. DNA ligase III discriminates against nicked substrates that contain increasing stretches of ribonucleotides. Two, but not five or more ribonucleotides, can be ligated. ( D ) As in C, except performed with T4 ligase (1–8 U). T4 ligase can ligate five but not 10 ribonucleotides. ( E ) T4 ligase-mediated ligation is abolished in the presence of the mutant RNase H1 proteins. The letters a-d correspond to the illustrations in panel A.
Techniques Used: DNA Synthesis, Ligation, Activity Assay, Mutagenesis
73) Product Images from "Evidence that base stacking potential in annealed 3' overhangs determines polymerase utilization in yeast nonhomologous end joining"
Article Title: Evidence that base stacking potential in annealed 3' overhangs determines polymerase utilization in yeast nonhomologous end joining
Journal:
doi: 10.1016/j.dnarep.2007.07.018
Figure Legend Snippet: 3’ dideoxynucleotide termini inhibit NHEJ. (A) Addition of the 3’ dideoxynucleotide was verified using pTW582, which forms compatible overhangs upon BstXI digestion. The linearized plasmid was incubated with T4 DNA ligase before (lane
Techniques Used: Non-Homologous End Joining, Plasmid Preparation, Incubation
74) Product Images from "MDP: A Deinococcus Mn2+-Decapeptide Complex Protects Mice from Ionizing Radiation"
Article Title: MDP: A Deinococcus Mn2+-Decapeptide Complex Protects Mice from Ionizing Radiation
Journal: PLoS ONE
doi: 10.1371/journal.pone.0160575
Figure Legend Snippet: Radioprotection of T4 DNA ligase by rationally-designed peptides. T4 DNA ligase was irradiated in the indicated mixtures and then tested for residual activity by incubation with linearized (L) pUC19 DNA (2.686 kbp). Formation of supercoiled (SC) and open-circular (OC) DNA forms were determined by agarose gel electrophoresis. Peptides were added to 25 mM Pi with or without 1 mM MnCl 2 to the following concentrations: DP1-L, 3 mM; DP1-D, 3 mM; DP2, 3 mM; OP1, 3.75 mM; HP1, 5 mM. Note, the final concentration of peptides in the reaction mixtures corresponded to 30 mM of total amino acid residues. Other abbreviations: Pi, potassium phosphate buffer (pH 7.4); M, DNA size standards (Mass Ruler DNA Ladder mix; Fermentas, Glen Burnie, MD).
Techniques Used: Irradiation, Activity Assay, Incubation, Agarose Gel Electrophoresis, Concentration Assay
75) Product Images from "Iron mediates catalysis of nucleic acid processing enzymes: support for Fe(II) as a cofactor before the great oxidation event"
Article Title: Iron mediates catalysis of nucleic acid processing enzymes: support for Fe(II) as a cofactor before the great oxidation event
Journal: Nucleic Acids Research
doi: 10.1093/nar/gkx171
Figure Legend Snippet: Fe 2+ can replace Mg 2+ as a cofactor for T4 DNA ligase. Ligation products of sliding-half complementary oligonucleotides are observed with both Mg 2+ and Fe 2+ . No product is observed in the divalent-minus negative control. See ‘Materials and Methods’ section for oligonucleotide sequences.
Techniques Used: Ligation, Negative Control
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Transduction:Article Title: TMEM120A and B: Nuclear Envelope Transmembrane Proteins Important for Adipocyte Differentiation Article Snippet: This lentiviral vector does not have a selection marker, but as lentiviruses were used to transduce cells the efficiencies should be high. .. The resulting PCR product was gel-purified and simultaneously phosphorylated and ligated using PNK and Clone Assay:Article Title: A comparison between the recombinant expression and chemical synthesis of a short cysteine-rich insecticidal spider peptide Article Snippet: Plasmids pCR®2.1-TOPO® (Invitrogen), pQE40 (Qiagen) and pET28a+ (Novagen) were used for cloning and production of the proteins linked to a 6His-tag. .. Restriction enzymes, Taq polymerase, factor Xa and Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation Article Snippet: The inserts and the vector were ligated in a 1 : 3 molar ratio using the Amplification:Article Title: Smc5/6 Antagonism by HBx Is an Evolutionarily Conserved Function of Hepatitis B Virus Infection in Mammals Article Snippet: The X insert was ligated to the pWPT-GFP backbone between the PstI and NotI sites following the T4 DNA (New England BioLabs [NEB]; Article Title: TMEM120A and B: Nuclear Envelope Transmembrane Proteins Important for Adipocyte Differentiation Article Snippet: For generating SBP constructs, SBP was amplified from the pTrAP vector [ ] and subcloned via BamHI and NotI sites into the pEGFP-N2 vector, replacing GFP with SBP. .. The resulting PCR product was gel-purified and simultaneously phosphorylated and ligated using PNK and Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation Article Snippet: After digestion of the three DNA fragments and the target vector (pGL3-basic, Promega) using the respective restriction enzymes (gBlock1: Xho I and Pst I, gBlock2: Pst I and Pvu I, PCR amplicon 3: Pvu I and Hind III, pGL3-basic: Xho I and Hind III, NEB, 90 min 37°C) the DNA fragments and the vector were purified using the Wizard SV Gel and PCR Clean-Up System (Promega) according manufacturer's instructions. .. The inserts and the vector were ligated in a 1 : 3 molar ratio using the DNA Ligation:Article Title: Gain of function mutant p53 proteins cooperate with E2F4 to transcriptionally downregulate RAD17 and BRCA1 gene expression Article Snippet: 200 ng of this DNA was incubated with nuclear extracts for 1h at 25°C in a reaction mixture containing 1 × ligase buffer and 1 μl of Synthesized:Article Title: Smc5/6 Antagonism by HBx Is an Evolutionarily Conserved Function of Hepatitis B Virus Infection in Mammals Article Snippet: The X coding regions (synthesized by Genewiz) from hepadnaviruses infecting the New World wooly monkey ( Lagothrix ) (WMHBx) and three distant bat species, including the roundleaf bat ( Hipposideros cf. ruber ), the horseshoe bat ( Rhinolophus Alcyone ), and the tent-making bat ( Uroderma bilobatum ) (RBHBx, HBHBx, TBHBx, respectively) , were expressed from the same vector. .. The X insert was ligated to the pWPT-GFP backbone between the PstI and NotI sites following the T4 DNA (New England BioLabs [NEB]; Article Title: Cell Differentiation in a Bacillus thuringiensis Population during Planktonic Growth, Biofilm Formation, and Host Infection Article Snippet: Restriction enzymes, Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation Article Snippet: Two fragments were synthesized (gBlock fragments-IDT), reaching from position chr15: 99589250-99589987 (gBlock1) and chr15: 99589970-99590606 (gBlock2) of the GRCm38/mm10 assembly and the third was obtained by PCR of region Chr15:99590527-99591271. .. The inserts and the vector were ligated in a 1 : 3 molar ratio using the Construct:Article Title: Cell Differentiation in a Bacillus thuringiensis Population during Planktonic Growth, Biofilm Formation, and Host Infection Article Snippet: Restriction enzymes, Article Title: TMEM120A and B: Nuclear Envelope Transmembrane Proteins Important for Adipocyte Differentiation Article Snippet: For generating SBP constructs, SBP was amplified from the pTrAP vector [ ] and subcloned via BamHI and NotI sites into the pEGFP-N2 vector, replacing GFP with SBP. .. The resulting PCR product was gel-purified and simultaneously phosphorylated and ligated using PNK and Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation Article Snippet: Promoter Transfection Experiments with Luciferase Vector A construct of the murine AQP5 promoter (2021 bp upstream of the ATG start codon) was produced by the fusion of three overlapping fragments treated with restriction enzymes followed by ligation. .. The inserts and the vector were ligated in a 1 : 3 molar ratio using the Electrophoresis:Article Title: Rbfox3 Controls the Biogenesis of a Subset of MicroRNAs Article Snippet: In brief, miRNAs were captured from 4 μg of total RNA using a bridge oligonucleotide that is designed to specifically detect one miRNA variant, and ligated to a 32 P-labeled detection oligonucleotide using Incubation:Article Title: MXS-Chaining: A Highly Efficient Cloning Platform for Imaging and Flow Cytometry Approaches in Mammalian Systems Article Snippet: .. Ligation For a typical ligation, 1 μ l of gel elution of the cut backbone, 8 μ l of gel elution of the cut insert, 1 μ l of Article Title: High-resolution TADs reveal DNA sequences underlying genome organization in flies Article Snippet: .. Ligase mix was added (1X Ligation buffer NEB B0202, 0.8% Triton X-100, 0.1 mg/ml BSA, 2000 U Article Title: Gain of function mutant p53 proteins cooperate with E2F4 to transcriptionally downregulate RAD17 and BRCA1 gene expression Article Snippet: .. 200 ng of this DNA was incubated with nuclear extracts for 1h at 25°C in a reaction mixture containing 1 × ligase buffer and 1 μl of Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation Article Snippet: .. The inserts and the vector were ligated in a 1 : 3 molar ratio using the Luciferase:Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation Article Snippet: Paragraph title: 2.8. Promoter Transfection Experiments with Luciferase Vector ... The inserts and the vector were ligated in a 1 : 3 molar ratio using the Expressing:Article Title: Smc5/6 Antagonism by HBx Is an Evolutionarily Conserved Function of Hepatitis B Virus Infection in Mammals Article Snippet: Paragraph title: Expression plasmids. ... The X insert was ligated to the pWPT-GFP backbone between the PstI and NotI sites following the T4 DNA (New England BioLabs [NEB]; Article Title: A comparison between the recombinant expression and chemical synthesis of a short cysteine-rich insecticidal spider peptide Article Snippet: M15 and BL21 were used for the expression of the proteins. .. Restriction enzymes, Taq polymerase, factor Xa and Transformation Assay:Article Title: Smc5/6 Antagonism by HBx Is an Evolutionarily Conserved Function of Hepatitis B Virus Infection in Mammals Article Snippet: The X insert was ligated to the pWPT-GFP backbone between the PstI and NotI sites following the T4 DNA (New England BioLabs [NEB]; Article Title: TMEM120A and B: Nuclear Envelope Transmembrane Proteins Important for Adipocyte Differentiation Article Snippet: .. The resulting PCR product was gel-purified and simultaneously phosphorylated and ligated using PNK and Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation Article Snippet: The inserts and the vector were ligated in a 1 : 3 molar ratio using the Activated Clotting Time Assay:Article Title: Rbfox3 Controls the Biogenesis of a Subset of MicroRNAs Article Snippet: In brief, miRNAs were captured from 4 μg of total RNA using a bridge oligonucleotide that is designed to specifically detect one miRNA variant, and ligated to a 32 P-labeled detection oligonucleotide using Countercurrent Chromatography:Article Title: Rbfox3 Controls the Biogenesis of a Subset of MicroRNAs Article Snippet: In brief, miRNAs were captured from 4 μg of total RNA using a bridge oligonucleotide that is designed to specifically detect one miRNA variant, and ligated to a 32 P-labeled detection oligonucleotide using Transfection:Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation Article Snippet: Paragraph title: 2.8. Promoter Transfection Experiments with Luciferase Vector ... The inserts and the vector were ligated in a 1 : 3 molar ratio using the Ligation:Article Title: MXS-Chaining: A Highly Efficient Cloning Platform for Imaging and Flow Cytometry Approaches in Mammalian Systems Article Snippet: .. Ligation For a typical ligation, 1 μ l of gel elution of the cut backbone, 8 μ l of gel elution of the cut insert, 1 μ l of Article Title: High-resolution TADs reveal DNA sequences underlying genome organization in flies Article Snippet: .. Ligase mix was added (1X Ligation buffer NEB B0202, 0.8% Triton X-100, 0.1 mg/ml BSA, 2000 U Article Title: Smc5/6 Antagonism by HBx Is an Evolutionarily Conserved Function of Hepatitis B Virus Infection in Mammals Article Snippet: .. The X insert was ligated to the pWPT-GFP backbone between the PstI and NotI sites following the T4 DNA (New England BioLabs [NEB]; Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation Article Snippet: Promoter Transfection Experiments with Luciferase Vector A construct of the murine AQP5 promoter (2021 bp upstream of the ATG start codon) was produced by the fusion of three overlapping fragments treated with restriction enzymes followed by ligation. .. The inserts and the vector were ligated in a 1 : 3 molar ratio using the Article Title: Assessing long-distance RNA sequence connectivity via RNA-templated DNA–DNA ligation Article Snippet: .. Final ligation conditions in were (left panel) 1.5 μM ssDNA or RNA template, 5′-32 P-labeled oligos (10 μM each), and 1 μl of neat indicated enzyme (specific units varied according to the manufacturer and enzyme) in manufacturer's recommended buffer; (right panel) 250 nM RNA template, 5′-32 P-labeled oligos (500 nM each), and 10 U/μl Rnl2 or 20 U/μl DNA Sequencing:Article Title: TMEM120A and B: Nuclear Envelope Transmembrane Proteins Important for Adipocyte Differentiation Article Snippet: The resulting PCR product was gel-purified and simultaneously phosphorylated and ligated using PNK and Polymerase Chain Reaction:Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq Article Snippet: KK8235) PEG/NaCl SPRI solution, supplied with HTP Library Preparation Kit PCR-free (96rxn) (Kapa Biosystems) 2× Kapa HiFi HotStart Ready Mix (Kapa Biosystems, cat.no. .. Article Title: Using specific length amplified fragment sequencing to construct the high-density genetic map for Vitis (Vitis vinifera L. × Vitis amurensis Rupr.) Article Snippet: The genomic DNA from each sample was treated with Rsa I, Hae III (NEB, Ipswich, MA, USA), Article Title: Cell Differentiation in a Bacillus thuringiensis Population during Planktonic Growth, Biofilm Formation, and Host Infection Article Snippet: PCR-amplified fragments and digested fragments separated on 0.8% agarose gels were purified with kits from Qiagen (France). .. Restriction enzymes, Article Title: A comparison between the recombinant expression and chemical synthesis of a short cysteine-rich insecticidal spider peptide Article Snippet: Plasmids pCR®2.1-TOPO® (Invitrogen), pQE40 (Qiagen) and pET28a+ (Novagen) were used for cloning and production of the proteins linked to a 6His-tag. .. Restriction enzymes, Taq polymerase, factor Xa and Article Title: TMEM120A and B: Nuclear Envelope Transmembrane Proteins Important for Adipocyte Differentiation Article Snippet: .. The resulting PCR product was gel-purified and simultaneously phosphorylated and ligated using PNK and Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation Article Snippet: After digestion of the three DNA fragments and the target vector (pGL3-basic, Promega) using the respective restriction enzymes (gBlock1: Xho I and Pst I, gBlock2: Pst I and Pvu I, PCR amplicon 3: Pvu I and Hind III, pGL3-basic: Xho I and Hind III, NEB, 90 min 37°C) the DNA fragments and the vector were purified using the Wizard SV Gel and PCR Clean-Up System (Promega) according manufacturer's instructions. .. The inserts and the vector were ligated in a 1 : 3 molar ratio using the Binding Assay:Article Title: High-resolution TADs reveal DNA sequences underlying genome organization in flies Article Snippet: Ligase mix was added (1X Ligation buffer NEB B0202, 0.8% Triton X-100, 0.1 mg/ml BSA, 2000 U Cellular Antioxidant Activity Assay:Article Title: Rbfox3 Controls the Biogenesis of a Subset of MicroRNAs Article Snippet: In brief, miRNAs were captured from 4 μg of total RNA using a bridge oligonucleotide that is designed to specifically detect one miRNA variant, and ligated to a 32 P-labeled detection oligonucleotide using Hi-C:Article Title: High-resolution TADs reveal DNA sequences underlying genome organization in flies Article Snippet: Paragraph title: In situ Hi-C of knockdown and control cells ... Ligase mix was added (1X Ligation buffer NEB B0202, 0.8% Triton X-100, 0.1 mg/ml BSA, 2000 U Marker:Article Title: Rbfox3 Controls the Biogenesis of a Subset of MicroRNAs Article Snippet: In brief, miRNAs were captured from 4 μg of total RNA using a bridge oligonucleotide that is designed to specifically detect one miRNA variant, and ligated to a 32 P-labeled detection oligonucleotide using Article Title: TMEM120A and B: Nuclear Envelope Transmembrane Proteins Important for Adipocyte Differentiation Article Snippet: This lentiviral vector does not have a selection marker, but as lentiviruses were used to transduce cells the efficiencies should be high. .. The resulting PCR product was gel-purified and simultaneously phosphorylated and ligated using PNK and Mutagenesis:Article Title: TMEM120A and B: Nuclear Envelope Transmembrane Proteins Important for Adipocyte Differentiation Article Snippet: To generate the ΔNTD mutant of human TMEM120A-SBP (lacking amino acids 2–105), site directed mutagenesis was done using the NEB Q5 mutagenesis kit strategy with minor modifications. .. The resulting PCR product was gel-purified and simultaneously phosphorylated and ligated using PNK and Subcloning:Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq Article Snippet: 28704) XL1-Blue subcloning grade competent cells (Agilent, cat.no. .. Size-exclusion Chromatography:Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation Article Snippet: The amplicon was produced with genomic DNA from EpH4 cells extracted using the Gentra Puregene Cell Kit (QIAGEN) according to manufacturer's instructions and amplified using 0.125 U OneTaq Hot Start DNA polymerase (NEB) with an initial heating step of 94°C for 5 min followed by 45 cycles at 94°C for 15 sec, 58°C for 15 sec, and 68°C for 45 sec (primer sequences are shown in Supplementary Table 1). .. The inserts and the vector were ligated in a 1 : 3 molar ratio using the Labeling:Article Title: Rbfox3 Controls the Biogenesis of a Subset of MicroRNAs Article Snippet: Splinted-ligation-mediated miRNA detection MiRNAs were detected using the miRtect-IT miRNA Labeling and Detection Kit (Affymetrix) according to the manufacturer’s instructions. .. In brief, miRNAs were captured from 4 μg of total RNA using a bridge oligonucleotide that is designed to specifically detect one miRNA variant, and ligated to a 32 P-labeled detection oligonucleotide using Purification:Article Title: Using specific length amplified fragment sequencing to construct the high-density genetic map for Vitis (Vitis vinifera L. × Vitis amurensis Rupr.) Article Snippet: The genomic DNA from each sample was treated with Rsa I, Hae III (NEB, Ipswich, MA, USA), Article Title: Cell Differentiation in a Bacillus thuringiensis Population during Planktonic Growth, Biofilm Formation, and Host Infection Article Snippet: PCR-amplified fragments and digested fragments separated on 0.8% agarose gels were purified with kits from Qiagen (France). .. Restriction enzymes, Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation Article Snippet: After digestion of the three DNA fragments and the target vector (pGL3-basic, Promega) using the respective restriction enzymes (gBlock1: Xho I and Pst I, gBlock2: Pst I and Pvu I, PCR amplicon 3: Pvu I and Hind III, pGL3-basic: Xho I and Hind III, NEB, 90 min 37°C) the DNA fragments and the vector were purified using the Wizard SV Gel and PCR Clean-Up System (Promega) according manufacturer's instructions. .. The inserts and the vector were ligated in a 1 : 3 molar ratio using the Sequencing:Article Title: Smc5/6 Antagonism by HBx Is an Evolutionarily Conserved Function of Hepatitis B Virus Infection in Mammals Article Snippet: The X insert was ligated to the pWPT-GFP backbone between the PstI and NotI sites following the T4 DNA (New England BioLabs [NEB]; Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation Article Snippet: The inserts and the vector were ligated in a 1 : 3 molar ratio using the Gel Extraction:Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq Article Snippet: R3535L) 10× CutSmart Buffer (New England BioLabs) supplied with Bsa I-HF QIAquick Gel extraction kit (Qiagen, cat.no. .. Chloramphenicol Acetyltransferase Assay:Article Title: Rbfox3 Controls the Biogenesis of a Subset of MicroRNAs Article Snippet: In brief, miRNAs were captured from 4 μg of total RNA using a bridge oligonucleotide that is designed to specifically detect one miRNA variant, and ligated to a 32 P-labeled detection oligonucleotide using Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq Article Snippet: .. Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq Article Snippet: .. Article Title: Smc5/6 Antagonism by HBx Is an Evolutionarily Conserved Function of Hepatitis B Virus Infection in Mammals Article Snippet: The X insert was ligated to the pWPT-GFP backbone between the PstI and NotI sites following the T4 DNA (New England BioLabs [NEB]; Plasmid Preparation:Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq Article Snippet: Article Title: Smc5/6 Antagonism by HBx Is an Evolutionarily Conserved Function of Hepatitis B Virus Infection in Mammals Article Snippet: The X coding regions (synthesized by Genewiz) from hepadnaviruses infecting the New World wooly monkey ( Lagothrix ) (WMHBx) and three distant bat species, including the roundleaf bat ( Hipposideros cf. ruber ), the horseshoe bat ( Rhinolophus Alcyone ), and the tent-making bat ( Uroderma bilobatum ) (RBHBx, HBHBx, TBHBx, respectively) , were expressed from the same vector. .. The X insert was ligated to the pWPT-GFP backbone between the PstI and NotI sites following the T4 DNA (New England BioLabs [NEB]; Article Title: Cell Differentiation in a Bacillus thuringiensis Population during Planktonic Growth, Biofilm Formation, and Host Infection Article Snippet: Plasmid DNA was extracted from E. coli by a standard alkaline lysis procedure with QIAprep spin columns (Qiagen, France). .. Restriction enzymes, Article Title: A comparison between the recombinant expression and chemical synthesis of a short cysteine-rich insecticidal spider peptide Article Snippet: Bacterial strains, enzymes and plasmids E. coli XL1 Blue was used for plasmid propagation. .. Restriction enzymes, Taq polymerase, factor Xa and Article Title: TMEM120A and B: Nuclear Envelope Transmembrane Proteins Important for Adipocyte Differentiation Article Snippet: Briefly, non-phosphorylated primers were used (forward 5’-GGATTGTACCTGAGCCTGGTTCTG and reverse 5’-CATGGGTCGAGATCTGAGTCCG ) in a PCR reaction with Phusion polymerase and TMEM120A-SBP plasmid. .. The resulting PCR product was gel-purified and simultaneously phosphorylated and ligated using PNK and Article Title: Gain of function mutant p53 proteins cooperate with E2F4 to transcriptionally downregulate RAD17 and BRCA1 gene expression Article Snippet: Linearized pUC19 DNA vector (Takara biotechnology, Dalia, CO., LTD) was prepared as previously described. .. 200 ng of this DNA was incubated with nuclear extracts for 1h at 25°C in a reaction mixture containing 1 × ligase buffer and 1 μl of Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation Article Snippet: .. The inserts and the vector were ligated in a 1 : 3 molar ratio using the In Situ:Article Title: High-resolution TADs reveal DNA sequences underlying genome organization in flies Article Snippet: Paragraph title: In situ Hi-C of knockdown and control cells ... Ligase mix was added (1X Ligation buffer NEB B0202, 0.8% Triton X-100, 0.1 mg/ml BSA, 2000 U Selection:Article Title: TMEM120A and B: Nuclear Envelope Transmembrane Proteins Important for Adipocyte Differentiation Article Snippet: This lentiviral vector does not have a selection marker, but as lentiviruses were used to transduce cells the efficiencies should be high. .. The resulting PCR product was gel-purified and simultaneously phosphorylated and ligated using PNK and Agarose Gel Electrophoresis:Article Title: Gain of function mutant p53 proteins cooperate with E2F4 to transcriptionally downregulate RAD17 and BRCA1 gene expression Article Snippet: 200 ng of this DNA was incubated with nuclear extracts for 1h at 25°C in a reaction mixture containing 1 × ligase buffer and 1 μl of In Vitro:Article Title: Gain of function mutant p53 proteins cooperate with E2F4 to transcriptionally downregulate RAD17 and BRCA1 gene expression Article Snippet: Paragraph title: T4 DNA ligase in vitro assay ... 200 ng of this DNA was incubated with nuclear extracts for 1h at 25°C in a reaction mixture containing 1 × ligase buffer and 1 μl of Ethanol Precipitation:Article Title: Gain of function mutant p53 proteins cooperate with E2F4 to transcriptionally downregulate RAD17 and BRCA1 gene expression Article Snippet: 200 ng of this DNA was incubated with nuclear extracts for 1h at 25°C in a reaction mixture containing 1 × ligase buffer and 1 μl of Next-Generation Sequencing:Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq Article Snippet: Paragraph title: CIRCLE-seq library preparation and NGS ... Produced:Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation Article Snippet: The amplicon was produced with genomic DNA from EpH4 cells extracted using the Gentra Puregene Cell Kit (QIAGEN) according to manufacturer's instructions and amplified using 0.125 U OneTaq Hot Start DNA polymerase (NEB) with an initial heating step of 94°C for 5 min followed by 45 cycles at 94°C for 15 sec, 58°C for 15 sec, and 68°C for 45 sec (primer sequences are shown in Supplementary Table 1). .. The inserts and the vector were ligated in a 1 : 3 molar ratio using the Concentration Assay:Article Title: High-resolution TADs reveal DNA sequences underlying genome organization in flies Article Snippet: After 10 min incubation at room temperature, SDS was quenched adding 1% Triton X-100 (final concentration) and 1X of NEBuffer 3.1 (NEB, B7203S). .. Ligase mix was added (1X Ligation buffer NEB B0202, 0.8% Triton X-100, 0.1 mg/ml BSA, 2000 U Alkaline Lysis:Article Title: Cell Differentiation in a Bacillus thuringiensis Population during Planktonic Growth, Biofilm Formation, and Host Infection Article Snippet: Plasmid DNA was extracted from E. coli by a standard alkaline lysis procedure with QIAprep spin columns (Qiagen, France). .. Restriction enzymes, CTG Assay:Article Title: Rbfox3 Controls the Biogenesis of a Subset of MicroRNAs Article Snippet: In brief, miRNAs were captured from 4 μg of total RNA using a bridge oligonucleotide that is designed to specifically detect one miRNA variant, and ligated to a 32 P-labeled detection oligonucleotide using Staining:Article Title: Gain of function mutant p53 proteins cooperate with E2F4 to transcriptionally downregulate RAD17 and BRCA1 gene expression Article Snippet: 200 ng of this DNA was incubated with nuclear extracts for 1h at 25°C in a reaction mixture containing 1 × ligase buffer and 1 μl of Variant Assay:Article Title: Rbfox3 Controls the Biogenesis of a Subset of MicroRNAs Article Snippet: .. In brief, miRNAs were captured from 4 μg of total RNA using a bridge oligonucleotide that is designed to specifically detect one miRNA variant, and ligated to a 32 P-labeled detection oligonucleotide using |