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    CutSmart Buffer
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    CutSmart Buffer 5 0 ml
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    1) Product Images from "Exonuclease combinations reduce noises in 3D genomics technologies"

    Article Title: Exonuclease combinations reduce noises in 3D genomics technologies

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa106

    C-technology application and preliminary assessment in Hi-C. ( A ) DNA library (572 ng) linear noise elimination for C-technologies, after proximal-ligation. M (left), 1 kb DNA ladder; 1: LRL, LRL used for linear noise elimination. 2: LRC, LRC used for linear noise elimination; 3: LIC, LIC used for linear noise elimination; 4: I+IIIC, I+IIIC used for linear noise elimination; C: Co, without exonucleases digestion; M (right): 100 bp DNA ladder. ( B ) The yield ratio of remaining DNA after four linear elimination treatments, which was consistent to (A). The remaining ratio (gray) and eliminated ratio (purple) are shown together in the bar chart. ( C ) A 40 ng library was given four exonuclease treatment combinations, along with addition of plasmid chaperon carrier, as indicated. M: 1 kb + 100 bp DNA ladder. 1′: LRLP1, LRL with P1 chaperon used for linear noise elimination; 2′: LRC with P1 chaperon used for linear noise elimination; 3′: LICP1, LIC with P1 chaperon used for linear noise elimination; 4′: I+IIICP1, I+IIIC with P1 chaperon used for linear noise elimination; C’: P1 chaperon added, without exonucleases digestion. ( D ) The yield ratio of remaining DNA, after four linear DNA elimination treatments, which was consistent to ( C ). ( E ) HiC-Pro filtering results of Hi-C with exonuclease elimination LIC, compared with standard Hi-C in Figure 1B . LIC-Hi-C had three experimental replicates (LIC-Hi-C 1; LIC-Hi-C 2; and LIC-Hi-C 3). HiC-Pro terms are as indicated in Supplemental S.3.
    Figure Legend Snippet: C-technology application and preliminary assessment in Hi-C. ( A ) DNA library (572 ng) linear noise elimination for C-technologies, after proximal-ligation. M (left), 1 kb DNA ladder; 1: LRL, LRL used for linear noise elimination. 2: LRC, LRC used for linear noise elimination; 3: LIC, LIC used for linear noise elimination; 4: I+IIIC, I+IIIC used for linear noise elimination; C: Co, without exonucleases digestion; M (right): 100 bp DNA ladder. ( B ) The yield ratio of remaining DNA after four linear elimination treatments, which was consistent to (A). The remaining ratio (gray) and eliminated ratio (purple) are shown together in the bar chart. ( C ) A 40 ng library was given four exonuclease treatment combinations, along with addition of plasmid chaperon carrier, as indicated. M: 1 kb + 100 bp DNA ladder. 1′: LRLP1, LRL with P1 chaperon used for linear noise elimination; 2′: LRC with P1 chaperon used for linear noise elimination; 3′: LICP1, LIC with P1 chaperon used for linear noise elimination; 4′: I+IIICP1, I+IIIC with P1 chaperon used for linear noise elimination; C’: P1 chaperon added, without exonucleases digestion. ( D ) The yield ratio of remaining DNA, after four linear DNA elimination treatments, which was consistent to ( C ). ( E ) HiC-Pro filtering results of Hi-C with exonuclease elimination LIC, compared with standard Hi-C in Figure 1B . LIC-Hi-C had three experimental replicates (LIC-Hi-C 1; LIC-Hi-C 2; and LIC-Hi-C 3). HiC-Pro terms are as indicated in Supplemental S.3.

    Techniques Used: Hi-C, Ligation, Plasmid Preparation

    Quantification effects for different combination treatments. ( A ) Results of every linear elimination treatment, measured by Qubit. Plasmid (250 ng) and linear DNA (250 ng) were mixed, as templates (X, Mixture). Linear DNA (500 ng) (3-L, LIC-Lin) was set as control treatments. Every treatment had three replicates. 1-X, LRL-Mixture; 2-X, LRC-Mixture; 3-X, LIC-Mixture; 4-X, I+III-Mixture; 5-X, VIII4-Mixture; 6-X, VIIIC-Mixture. ( B ) The qPCR results from three exonuclease combination treatments (in accordance with Figure 2B ). The primer BH2 was used across the plasmid BamHI site. Same quality (SQ): The Qubit amounts of qPCR input for all treatments were consistent (Supplemental S.1.2). Same volume (SV): The qPCR input volumes were consistent (Supplemental S.1.4 and Supplementary Figure S5 ). P, pGL4.23 plasmid; L, linearized plasmid; X, Mixture; 3-P, LIC to cut plasmid; 3-L, LIC to cut linearized plasmid. Three experiments for each treatment were combined for enlarging the volume before purification. ( C ) The qPCR results correspond with Figure 2C . The primer was BH2. Data are presented as mean ± SEM.
    Figure Legend Snippet: Quantification effects for different combination treatments. ( A ) Results of every linear elimination treatment, measured by Qubit. Plasmid (250 ng) and linear DNA (250 ng) were mixed, as templates (X, Mixture). Linear DNA (500 ng) (3-L, LIC-Lin) was set as control treatments. Every treatment had three replicates. 1-X, LRL-Mixture; 2-X, LRC-Mixture; 3-X, LIC-Mixture; 4-X, I+III-Mixture; 5-X, VIII4-Mixture; 6-X, VIIIC-Mixture. ( B ) The qPCR results from three exonuclease combination treatments (in accordance with Figure 2B ). The primer BH2 was used across the plasmid BamHI site. Same quality (SQ): The Qubit amounts of qPCR input for all treatments were consistent (Supplemental S.1.2). Same volume (SV): The qPCR input volumes were consistent (Supplemental S.1.4 and Supplementary Figure S5 ). P, pGL4.23 plasmid; L, linearized plasmid; X, Mixture; 3-P, LIC to cut plasmid; 3-L, LIC to cut linearized plasmid. Three experiments for each treatment were combined for enlarging the volume before purification. ( C ) The qPCR results correspond with Figure 2C . The primer was BH2. Data are presented as mean ± SEM.

    Techniques Used: Plasmid Preparation, Real-time Polymerase Chain Reaction, Purification

    Experimental results for exonuclease combinations treatment. ( A ) The cleavage mechanism of Lamada and Exonuclease I combinations. Other exonuclease combinations (Lambda and RecJF; Exonuclease I and Exonuclease III) are shown in Supplementary Figure S1A and Supplementary Table S1 . ( B ) Three exonuclease combinations (LRL; LRC; LIC) removed linear DNA from a paradigm mixture. M, 1 kb DNA ladder; P, pGL4.23 plasmid; L, linearized plasmid; X, mixture (plasmid and linear DNA 1:1); 1-X, LRL-Mixture, LRL to cut mixture; 2-X, LRC-Mixture, LRC to cut mixture; 3-X, LIC-Mixture, LIC to cut mixture; 3-P, LIC to cut plasmid; 3-L, LIC-Lin, LIC to cut linearized plasmid; MS, supercoiled ladder. ( C ) I+III combination and Exonuclease VIII, truncated elimination tests. 5-X, VIII4-Mixture, Exonuclease VIII, truncated within Buffer 4 to remove mixture; 6-X, VIIIC-Mixture, Exonuclease VIII, truncated within CutSmart buffer to remove mixture; 4-X: I+III-Mixture, I+III to remove mixture. Loading samples for agarose gel electrophoresis were purified by phenol-chloroform.
    Figure Legend Snippet: Experimental results for exonuclease combinations treatment. ( A ) The cleavage mechanism of Lamada and Exonuclease I combinations. Other exonuclease combinations (Lambda and RecJF; Exonuclease I and Exonuclease III) are shown in Supplementary Figure S1A and Supplementary Table S1 . ( B ) Three exonuclease combinations (LRL; LRC; LIC) removed linear DNA from a paradigm mixture. M, 1 kb DNA ladder; P, pGL4.23 plasmid; L, linearized plasmid; X, mixture (plasmid and linear DNA 1:1); 1-X, LRL-Mixture, LRL to cut mixture; 2-X, LRC-Mixture, LRC to cut mixture; 3-X, LIC-Mixture, LIC to cut mixture; 3-P, LIC to cut plasmid; 3-L, LIC-Lin, LIC to cut linearized plasmid; MS, supercoiled ladder. ( C ) I+III combination and Exonuclease VIII, truncated elimination tests. 5-X, VIII4-Mixture, Exonuclease VIII, truncated within Buffer 4 to remove mixture; 6-X, VIIIC-Mixture, Exonuclease VIII, truncated within CutSmart buffer to remove mixture; 4-X: I+III-Mixture, I+III to remove mixture. Loading samples for agarose gel electrophoresis were purified by phenol-chloroform.

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Purification

    Related Articles

    Isolation:

    Article Title: Depurination of colibactin-derived interstrand cross-links
    Article Snippet: Plasmid Linearization Test The pUC19 DNA isolated from plasmid cleavage assays with E. coli. was used for the plasmid linearization test. .. To set up the linearization reactions, 20 units of EcoRI-HF® (New England Biolabs®) was mixed with 500 ng of isolated DNA (40 units/µg DNA) in CutSmart® buffer (New England Biolabs®), pH 7.9, in a total volume of 50 µL for 30 min at 37 °C. .. The CutSmart® buffer (New England Biolabs®) contains 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, and 100 µg/mL BSA.

    Amplification:

    Article Title: Droplet Tn-Seq combines microfluidics with Tn-Seq for identifying complex single-cell phenotypes
    Article Snippet: Next 1× BSA (NEB), and two units of phi29 (0.57 U/ml), were added to the reaction, and incubated for 7 h at 30 °C, 10 min at 65 °C, and held at 4 °C. (2) In total, 10 μl of magnetic beads (Axygen – AxyPrep Mag PCR Clean-up Kit, MAGPCRCL50) were mixed with 30 μl of freshly made polyethylene glycol (PEG) solution (20% PEG 8000, 2.5 M NaCl, 10 mM Tris-base, 1 mM EDTA, 0.05% tween20, pH 8) and added to the 30 μl of sample, mixed, and incubated at room temperature for 20 min. A magnet was used to separate the bead/DNA complex from the PEG solution, and the beads were washed three times in 200 μl 70% ethanol (all magnetic bead washes were performed this way). .. Beads were then dried for 3 min at room temperature, and DNA was eluted off the beads with 12.7 μl of dH2 O. (3) In all, 11.49 μl of phi29 amplified DNA was then added to a MmeI digestion mix (two units NEB MmeI enzyme, 50 μM SAM, 1× CutSmart Buffer) in a total volume of 20 μl, and incubated for 2.5 h at 37 °C followed by 20 min at 65 °C. (4) In all, 1 μl of alkaline phosphatase (NEB - M0290S Calf Intestinal, CIP) was added to the sample and incubated for 1 h at 37 °C. (5) In total, 10 μl of magnetic beads plus 20 μl PEG solution per sample were used to wash the sample followed by elution in 14.3 μl of dH2 O. (6) T4 DNA ligase (NEB M0202L) was used to ligate DNA adapter barcodes by adding 13.12 μl DNA to 1 μl of 1:5 diluted adapter, 1× T4 DNA Ligase Reaction Buffer, and 400 units T4 DNA ligase, followed by incubation at 16 °C for 16 h, 65 °C for 10 min, and held at 10 °C. (7) In all, 10 μl magnetic beads plus 20 μl PEG solution were used to wash the sample followed by elution in 36 μl of dH2 O. (8) Adapter ligated DNA was then PCR amplified using Q5 high-fidelity DNA polymerase (NEB – M0491L) by adding 34 μl of DNA to 1X Q5 reaction buffer, 10 mM dNTPs, 0.45 μM of each primer (P1-M6-GAT-MmeI; P2-ADPT-Tnseq-primer; Supplementary Data ), one unit Q5 DNA polymerase, and incubated at 98 °C for 30 s, and 18–22 cycles of 98 °C for 10 s, 62 °C for 30 s, 72 °C for 15 s, followed by 72 °C for 2 min, and a 10 °C hold. (9) PCR products were gel purified and sequenced on an Illumina NextSeq 500 according to the manufacturer's protocol. .. Sequence analysis was performed with a series of in-house scripts , , .

    Incubation:

    Article Title: Droplet Tn-Seq combines microfluidics with Tn-Seq for identifying complex single-cell phenotypes
    Article Snippet: Next 1× BSA (NEB), and two units of phi29 (0.57 U/ml), were added to the reaction, and incubated for 7 h at 30 °C, 10 min at 65 °C, and held at 4 °C. (2) In total, 10 μl of magnetic beads (Axygen – AxyPrep Mag PCR Clean-up Kit, MAGPCRCL50) were mixed with 30 μl of freshly made polyethylene glycol (PEG) solution (20% PEG 8000, 2.5 M NaCl, 10 mM Tris-base, 1 mM EDTA, 0.05% tween20, pH 8) and added to the 30 μl of sample, mixed, and incubated at room temperature for 20 min. A magnet was used to separate the bead/DNA complex from the PEG solution, and the beads were washed three times in 200 μl 70% ethanol (all magnetic bead washes were performed this way). .. Beads were then dried for 3 min at room temperature, and DNA was eluted off the beads with 12.7 μl of dH2 O. (3) In all, 11.49 μl of phi29 amplified DNA was then added to a MmeI digestion mix (two units NEB MmeI enzyme, 50 μM SAM, 1× CutSmart Buffer) in a total volume of 20 μl, and incubated for 2.5 h at 37 °C followed by 20 min at 65 °C. (4) In all, 1 μl of alkaline phosphatase (NEB - M0290S Calf Intestinal, CIP) was added to the sample and incubated for 1 h at 37 °C. (5) In total, 10 μl of magnetic beads plus 20 μl PEG solution per sample were used to wash the sample followed by elution in 14.3 μl of dH2 O. (6) T4 DNA ligase (NEB M0202L) was used to ligate DNA adapter barcodes by adding 13.12 μl DNA to 1 μl of 1:5 diluted adapter, 1× T4 DNA Ligase Reaction Buffer, and 400 units T4 DNA ligase, followed by incubation at 16 °C for 16 h, 65 °C for 10 min, and held at 10 °C. (7) In all, 10 μl magnetic beads plus 20 μl PEG solution were used to wash the sample followed by elution in 36 μl of dH2 O. (8) Adapter ligated DNA was then PCR amplified using Q5 high-fidelity DNA polymerase (NEB – M0491L) by adding 34 μl of DNA to 1X Q5 reaction buffer, 10 mM dNTPs, 0.45 μM of each primer (P1-M6-GAT-MmeI; P2-ADPT-Tnseq-primer; Supplementary Data ), one unit Q5 DNA polymerase, and incubated at 98 °C for 30 s, and 18–22 cycles of 98 °C for 10 s, 62 °C for 30 s, 72 °C for 15 s, followed by 72 °C for 2 min, and a 10 °C hold. (9) PCR products were gel purified and sequenced on an Illumina NextSeq 500 according to the manufacturer's protocol. .. Sequence analysis was performed with a series of in-house scripts , , .

    Magnetic Beads:

    Article Title: Droplet Tn-Seq combines microfluidics with Tn-Seq for identifying complex single-cell phenotypes
    Article Snippet: Next 1× BSA (NEB), and two units of phi29 (0.57 U/ml), were added to the reaction, and incubated for 7 h at 30 °C, 10 min at 65 °C, and held at 4 °C. (2) In total, 10 μl of magnetic beads (Axygen – AxyPrep Mag PCR Clean-up Kit, MAGPCRCL50) were mixed with 30 μl of freshly made polyethylene glycol (PEG) solution (20% PEG 8000, 2.5 M NaCl, 10 mM Tris-base, 1 mM EDTA, 0.05% tween20, pH 8) and added to the 30 μl of sample, mixed, and incubated at room temperature for 20 min. A magnet was used to separate the bead/DNA complex from the PEG solution, and the beads were washed three times in 200 μl 70% ethanol (all magnetic bead washes were performed this way). .. Beads were then dried for 3 min at room temperature, and DNA was eluted off the beads with 12.7 μl of dH2 O. (3) In all, 11.49 μl of phi29 amplified DNA was then added to a MmeI digestion mix (two units NEB MmeI enzyme, 50 μM SAM, 1× CutSmart Buffer) in a total volume of 20 μl, and incubated for 2.5 h at 37 °C followed by 20 min at 65 °C. (4) In all, 1 μl of alkaline phosphatase (NEB - M0290S Calf Intestinal, CIP) was added to the sample and incubated for 1 h at 37 °C. (5) In total, 10 μl of magnetic beads plus 20 μl PEG solution per sample were used to wash the sample followed by elution in 14.3 μl of dH2 O. (6) T4 DNA ligase (NEB M0202L) was used to ligate DNA adapter barcodes by adding 13.12 μl DNA to 1 μl of 1:5 diluted adapter, 1× T4 DNA Ligase Reaction Buffer, and 400 units T4 DNA ligase, followed by incubation at 16 °C for 16 h, 65 °C for 10 min, and held at 10 °C. (7) In all, 10 μl magnetic beads plus 20 μl PEG solution were used to wash the sample followed by elution in 36 μl of dH2 O. (8) Adapter ligated DNA was then PCR amplified using Q5 high-fidelity DNA polymerase (NEB – M0491L) by adding 34 μl of DNA to 1X Q5 reaction buffer, 10 mM dNTPs, 0.45 μM of each primer (P1-M6-GAT-MmeI; P2-ADPT-Tnseq-primer; Supplementary Data ), one unit Q5 DNA polymerase, and incubated at 98 °C for 30 s, and 18–22 cycles of 98 °C for 10 s, 62 °C for 30 s, 72 °C for 15 s, followed by 72 °C for 2 min, and a 10 °C hold. (9) PCR products were gel purified and sequenced on an Illumina NextSeq 500 according to the manufacturer's protocol. .. Sequence analysis was performed with a series of in-house scripts , , .

    Polymerase Chain Reaction:

    Article Title: Droplet Tn-Seq combines microfluidics with Tn-Seq for identifying complex single-cell phenotypes
    Article Snippet: Next 1× BSA (NEB), and two units of phi29 (0.57 U/ml), were added to the reaction, and incubated for 7 h at 30 °C, 10 min at 65 °C, and held at 4 °C. (2) In total, 10 μl of magnetic beads (Axygen – AxyPrep Mag PCR Clean-up Kit, MAGPCRCL50) were mixed with 30 μl of freshly made polyethylene glycol (PEG) solution (20% PEG 8000, 2.5 M NaCl, 10 mM Tris-base, 1 mM EDTA, 0.05% tween20, pH 8) and added to the 30 μl of sample, mixed, and incubated at room temperature for 20 min. A magnet was used to separate the bead/DNA complex from the PEG solution, and the beads were washed three times in 200 μl 70% ethanol (all magnetic bead washes were performed this way). .. Beads were then dried for 3 min at room temperature, and DNA was eluted off the beads with 12.7 μl of dH2 O. (3) In all, 11.49 μl of phi29 amplified DNA was then added to a MmeI digestion mix (two units NEB MmeI enzyme, 50 μM SAM, 1× CutSmart Buffer) in a total volume of 20 μl, and incubated for 2.5 h at 37 °C followed by 20 min at 65 °C. (4) In all, 1 μl of alkaline phosphatase (NEB - M0290S Calf Intestinal, CIP) was added to the sample and incubated for 1 h at 37 °C. (5) In total, 10 μl of magnetic beads plus 20 μl PEG solution per sample were used to wash the sample followed by elution in 14.3 μl of dH2 O. (6) T4 DNA ligase (NEB M0202L) was used to ligate DNA adapter barcodes by adding 13.12 μl DNA to 1 μl of 1:5 diluted adapter, 1× T4 DNA Ligase Reaction Buffer, and 400 units T4 DNA ligase, followed by incubation at 16 °C for 16 h, 65 °C for 10 min, and held at 10 °C. (7) In all, 10 μl magnetic beads plus 20 μl PEG solution were used to wash the sample followed by elution in 36 μl of dH2 O. (8) Adapter ligated DNA was then PCR amplified using Q5 high-fidelity DNA polymerase (NEB – M0491L) by adding 34 μl of DNA to 1X Q5 reaction buffer, 10 mM dNTPs, 0.45 μM of each primer (P1-M6-GAT-MmeI; P2-ADPT-Tnseq-primer; Supplementary Data ), one unit Q5 DNA polymerase, and incubated at 98 °C for 30 s, and 18–22 cycles of 98 °C for 10 s, 62 °C for 30 s, 72 °C for 15 s, followed by 72 °C for 2 min, and a 10 °C hold. (9) PCR products were gel purified and sequenced on an Illumina NextSeq 500 according to the manufacturer's protocol. .. Sequence analysis was performed with a series of in-house scripts , , .

    Article Title: A simple and efficient cloning system for CRISPR/Cas9-mediated genome editing in rice
    Article Snippet: Primers containing adaptors for Golden Gate cloning (OJH307 and OJH308, ) were used in the amplification with PJG090 as the template. .. The reagents were recommended as following: one ul of PCR product, 50 ng of PJG112, 1 ul of Cutsmart Buffer (NEB), 0.4 ul of T4 ligase buffer (NEB), 5 U of Bsa I (NEB), 20 U of T4 DNA ligase (NEB) and add ddH2 O to 10 ul. ..

    Purification:

    Article Title: Droplet Tn-Seq combines microfluidics with Tn-Seq for identifying complex single-cell phenotypes
    Article Snippet: Next 1× BSA (NEB), and two units of phi29 (0.57 U/ml), were added to the reaction, and incubated for 7 h at 30 °C, 10 min at 65 °C, and held at 4 °C. (2) In total, 10 μl of magnetic beads (Axygen – AxyPrep Mag PCR Clean-up Kit, MAGPCRCL50) were mixed with 30 μl of freshly made polyethylene glycol (PEG) solution (20% PEG 8000, 2.5 M NaCl, 10 mM Tris-base, 1 mM EDTA, 0.05% tween20, pH 8) and added to the 30 μl of sample, mixed, and incubated at room temperature for 20 min. A magnet was used to separate the bead/DNA complex from the PEG solution, and the beads were washed three times in 200 μl 70% ethanol (all magnetic bead washes were performed this way). .. Beads were then dried for 3 min at room temperature, and DNA was eluted off the beads with 12.7 μl of dH2 O. (3) In all, 11.49 μl of phi29 amplified DNA was then added to a MmeI digestion mix (two units NEB MmeI enzyme, 50 μM SAM, 1× CutSmart Buffer) in a total volume of 20 μl, and incubated for 2.5 h at 37 °C followed by 20 min at 65 °C. (4) In all, 1 μl of alkaline phosphatase (NEB - M0290S Calf Intestinal, CIP) was added to the sample and incubated for 1 h at 37 °C. (5) In total, 10 μl of magnetic beads plus 20 μl PEG solution per sample were used to wash the sample followed by elution in 14.3 μl of dH2 O. (6) T4 DNA ligase (NEB M0202L) was used to ligate DNA adapter barcodes by adding 13.12 μl DNA to 1 μl of 1:5 diluted adapter, 1× T4 DNA Ligase Reaction Buffer, and 400 units T4 DNA ligase, followed by incubation at 16 °C for 16 h, 65 °C for 10 min, and held at 10 °C. (7) In all, 10 μl magnetic beads plus 20 μl PEG solution were used to wash the sample followed by elution in 36 μl of dH2 O. (8) Adapter ligated DNA was then PCR amplified using Q5 high-fidelity DNA polymerase (NEB – M0491L) by adding 34 μl of DNA to 1X Q5 reaction buffer, 10 mM dNTPs, 0.45 μM of each primer (P1-M6-GAT-MmeI; P2-ADPT-Tnseq-primer; Supplementary Data ), one unit Q5 DNA polymerase, and incubated at 98 °C for 30 s, and 18–22 cycles of 98 °C for 10 s, 62 °C for 30 s, 72 °C for 15 s, followed by 72 °C for 2 min, and a 10 °C hold. (9) PCR products were gel purified and sequenced on an Illumina NextSeq 500 according to the manufacturer's protocol. .. Sequence analysis was performed with a series of in-house scripts , , .

    Plasmid Preparation:

    Article Title: Restriction Endonucleases from Invasive Neisseria gonorrhoeae Cause Double-Strand Breaks and Distort Mitosis in Epithelial Cells during Infection
    Article Snippet: .. Portions of lysates were further heat inactivated (HI) at 95°C for 10 min. One µg of the commercial plasmid pECFP-N1 (Clonetech, CA, USA) was subjected to either MS11 P+ lysate or HI lysate together with CutSmart buffer (New England Biolabs, Ipswich, MA, USA) for 1 h. As controls, circular/uncut pECFP-N1 was used as well as HindIII (Roche, Mannheim, Germany) linearized pECFP-N1. .. The plasmid reactions were run on 1% agarose gel electrophoresis in 1xTBE buffer and stained with ethidium bromide.

    Article Title: Exonuclease combinations reduce noises in 3D genomics technologies
    Article Snippet: Exonuclease combination pre-tests for linearized DNA elimination The pGL4.23 (4283 bp, P1) ( ) and pCDNA3.1-NLS-NgAgo (8246 bp, P2) plasmids were extracted, using Endo-free Plasmid Mini Kit II (E.Z.N.A.® , D6950). .. Linearized plasmid DNA was obtained in the following 200 μl solution/per reaction, including 23.4 μl plasmid, 5 μl BamHI-HF (HindIII for P2) (NEB), 20 μl 10× Cutsmart Buffer (NEBuffer 2 for P2) and 151.6 μl H2 O. .. Three reactions were performed at 37°C for 1 h. To increase the yield of linearized plasmid DNA, three digested solutions were combined, followed by addition of 1.2-fold phenol/chloroform/isoamyl alcohol for DNA extraction ( ).

    Real-time Polymerase Chain Reaction:

    Article Title: Exonuclease combinations reduce noises in 3D genomics technologies
    Article Snippet: Agarose gel electrophoresis and real-time qPCR For agarose gel electrophoresis, loading samples (40 ng) were used for electrophoresis (0.8% agarose gel), performed at 100 V for 40 min. Gels were digitally imaged using a system adapted for photography (Bio-Rad Laboratories, CA, USA). .. After LRL (Lambda and RecJF within Lambda buffer) (NEB), LRC (Lambda and RecJF within Cutsmart buffer) (NEB), LIC (Lambda and Exonuclease I within Cutsmart buffer) (NEB) and I+IIIC (I+III or IIIIC) (Exonuclease I and Exonuclease III within Cutsmart buffer) (NEB) elimination, qPCR was performed. .. Primer BH2 for qPCR was designed across both ends of the BamHI restriction site.

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    New England Biolabs lrc
    C-technology application and preliminary assessment in Hi-C. ( A ) DNA library (572 ng) linear noise elimination for C-technologies, after proximal-ligation. M (left), 1 kb DNA ladder; 1: <t>LRL,</t> LRL used for linear noise elimination. 2: <t>LRC,</t> LRC used for linear noise elimination; 3: LIC, LIC used for linear noise elimination; 4: I+IIIC, I+IIIC used for linear noise elimination; C: Co, without exonucleases digestion; M (right): 100 bp DNA ladder. ( B ) The yield ratio of remaining DNA after four linear elimination treatments, which was consistent to (A). The remaining ratio (gray) and eliminated ratio (purple) are shown together in the bar chart. ( C ) A 40 ng library was given four exonuclease treatment combinations, along with addition of plasmid chaperon carrier, as indicated. M: 1 kb + 100 bp DNA ladder. 1′: LRLP1, LRL with P1 chaperon used for linear noise elimination; 2′: LRC with P1 chaperon used for linear noise elimination; 3′: LICP1, LIC with P1 chaperon used for linear noise elimination; 4′: I+IIICP1, I+IIIC with P1 chaperon used for linear noise elimination; C’: P1 chaperon added, without exonucleases digestion. ( D ) The yield ratio of remaining DNA, after four linear DNA elimination treatments, which was consistent to ( C ). ( E ) HiC-Pro filtering results of Hi-C with exonuclease elimination LIC, compared with standard Hi-C in Figure 1B . LIC-Hi-C had three experimental replicates (LIC-Hi-C 1; LIC-Hi-C 2; and LIC-Hi-C 3). HiC-Pro terms are as indicated in Supplemental S.3.
    Lrc, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    C-technology application and preliminary assessment in Hi-C. ( A ) DNA library (572 ng) linear noise elimination for C-technologies, after proximal-ligation. M (left), 1 kb DNA ladder; 1: LRL, LRL used for linear noise elimination. 2: LRC, LRC used for linear noise elimination; 3: LIC, LIC used for linear noise elimination; 4: I+IIIC, I+IIIC used for linear noise elimination; C: Co, without exonucleases digestion; M (right): 100 bp DNA ladder. ( B ) The yield ratio of remaining DNA after four linear elimination treatments, which was consistent to (A). The remaining ratio (gray) and eliminated ratio (purple) are shown together in the bar chart. ( C ) A 40 ng library was given four exonuclease treatment combinations, along with addition of plasmid chaperon carrier, as indicated. M: 1 kb + 100 bp DNA ladder. 1′: LRLP1, LRL with P1 chaperon used for linear noise elimination; 2′: LRC with P1 chaperon used for linear noise elimination; 3′: LICP1, LIC with P1 chaperon used for linear noise elimination; 4′: I+IIICP1, I+IIIC with P1 chaperon used for linear noise elimination; C’: P1 chaperon added, without exonucleases digestion. ( D ) The yield ratio of remaining DNA, after four linear DNA elimination treatments, which was consistent to ( C ). ( E ) HiC-Pro filtering results of Hi-C with exonuclease elimination LIC, compared with standard Hi-C in Figure 1B . LIC-Hi-C had three experimental replicates (LIC-Hi-C 1; LIC-Hi-C 2; and LIC-Hi-C 3). HiC-Pro terms are as indicated in Supplemental S.3.

    Journal: Nucleic Acids Research

    Article Title: Exonuclease combinations reduce noises in 3D genomics technologies

    doi: 10.1093/nar/gkaa106

    Figure Lengend Snippet: C-technology application and preliminary assessment in Hi-C. ( A ) DNA library (572 ng) linear noise elimination for C-technologies, after proximal-ligation. M (left), 1 kb DNA ladder; 1: LRL, LRL used for linear noise elimination. 2: LRC, LRC used for linear noise elimination; 3: LIC, LIC used for linear noise elimination; 4: I+IIIC, I+IIIC used for linear noise elimination; C: Co, without exonucleases digestion; M (right): 100 bp DNA ladder. ( B ) The yield ratio of remaining DNA after four linear elimination treatments, which was consistent to (A). The remaining ratio (gray) and eliminated ratio (purple) are shown together in the bar chart. ( C ) A 40 ng library was given four exonuclease treatment combinations, along with addition of plasmid chaperon carrier, as indicated. M: 1 kb + 100 bp DNA ladder. 1′: LRLP1, LRL with P1 chaperon used for linear noise elimination; 2′: LRC with P1 chaperon used for linear noise elimination; 3′: LICP1, LIC with P1 chaperon used for linear noise elimination; 4′: I+IIICP1, I+IIIC with P1 chaperon used for linear noise elimination; C’: P1 chaperon added, without exonucleases digestion. ( D ) The yield ratio of remaining DNA, after four linear DNA elimination treatments, which was consistent to ( C ). ( E ) HiC-Pro filtering results of Hi-C with exonuclease elimination LIC, compared with standard Hi-C in Figure 1B . LIC-Hi-C had three experimental replicates (LIC-Hi-C 1; LIC-Hi-C 2; and LIC-Hi-C 3). HiC-Pro terms are as indicated in Supplemental S.3.

    Article Snippet: After LRL (Lambda and RecJF within Lambda buffer) (NEB), LRC (Lambda and RecJF within Cutsmart buffer) (NEB), LIC (Lambda and Exonuclease I within Cutsmart buffer) (NEB) and I+IIIC (I+III or IIIIC) (Exonuclease I and Exonuclease III within Cutsmart buffer) (NEB) elimination, qPCR was performed.

    Techniques: Hi-C, Ligation, Plasmid Preparation

    Quantification effects for different combination treatments. ( A ) Results of every linear elimination treatment, measured by Qubit. Plasmid (250 ng) and linear DNA (250 ng) were mixed, as templates (X, Mixture). Linear DNA (500 ng) (3-L, LIC-Lin) was set as control treatments. Every treatment had three replicates. 1-X, LRL-Mixture; 2-X, LRC-Mixture; 3-X, LIC-Mixture; 4-X, I+III-Mixture; 5-X, VIII4-Mixture; 6-X, VIIIC-Mixture. ( B ) The qPCR results from three exonuclease combination treatments (in accordance with Figure 2B ). The primer BH2 was used across the plasmid BamHI site. Same quality (SQ): The Qubit amounts of qPCR input for all treatments were consistent (Supplemental S.1.2). Same volume (SV): The qPCR input volumes were consistent (Supplemental S.1.4 and Supplementary Figure S5 ). P, pGL4.23 plasmid; L, linearized plasmid; X, Mixture; 3-P, LIC to cut plasmid; 3-L, LIC to cut linearized plasmid. Three experiments for each treatment were combined for enlarging the volume before purification. ( C ) The qPCR results correspond with Figure 2C . The primer was BH2. Data are presented as mean ± SEM.

    Journal: Nucleic Acids Research

    Article Title: Exonuclease combinations reduce noises in 3D genomics technologies

    doi: 10.1093/nar/gkaa106

    Figure Lengend Snippet: Quantification effects for different combination treatments. ( A ) Results of every linear elimination treatment, measured by Qubit. Plasmid (250 ng) and linear DNA (250 ng) were mixed, as templates (X, Mixture). Linear DNA (500 ng) (3-L, LIC-Lin) was set as control treatments. Every treatment had three replicates. 1-X, LRL-Mixture; 2-X, LRC-Mixture; 3-X, LIC-Mixture; 4-X, I+III-Mixture; 5-X, VIII4-Mixture; 6-X, VIIIC-Mixture. ( B ) The qPCR results from three exonuclease combination treatments (in accordance with Figure 2B ). The primer BH2 was used across the plasmid BamHI site. Same quality (SQ): The Qubit amounts of qPCR input for all treatments were consistent (Supplemental S.1.2). Same volume (SV): The qPCR input volumes were consistent (Supplemental S.1.4 and Supplementary Figure S5 ). P, pGL4.23 plasmid; L, linearized plasmid; X, Mixture; 3-P, LIC to cut plasmid; 3-L, LIC to cut linearized plasmid. Three experiments for each treatment were combined for enlarging the volume before purification. ( C ) The qPCR results correspond with Figure 2C . The primer was BH2. Data are presented as mean ± SEM.

    Article Snippet: After LRL (Lambda and RecJF within Lambda buffer) (NEB), LRC (Lambda and RecJF within Cutsmart buffer) (NEB), LIC (Lambda and Exonuclease I within Cutsmart buffer) (NEB) and I+IIIC (I+III or IIIIC) (Exonuclease I and Exonuclease III within Cutsmart buffer) (NEB) elimination, qPCR was performed.

    Techniques: Plasmid Preparation, Real-time Polymerase Chain Reaction, Purification

    Experimental results for exonuclease combinations treatment. ( A ) The cleavage mechanism of Lamada and Exonuclease I combinations. Other exonuclease combinations (Lambda and RecJF; Exonuclease I and Exonuclease III) are shown in Supplementary Figure S1A and Supplementary Table S1 . ( B ) Three exonuclease combinations (LRL; LRC; LIC) removed linear DNA from a paradigm mixture. M, 1 kb DNA ladder; P, pGL4.23 plasmid; L, linearized plasmid; X, mixture (plasmid and linear DNA 1:1); 1-X, LRL-Mixture, LRL to cut mixture; 2-X, LRC-Mixture, LRC to cut mixture; 3-X, LIC-Mixture, LIC to cut mixture; 3-P, LIC to cut plasmid; 3-L, LIC-Lin, LIC to cut linearized plasmid; MS, supercoiled ladder. ( C ) I+III combination and Exonuclease VIII, truncated elimination tests. 5-X, VIII4-Mixture, Exonuclease VIII, truncated within Buffer 4 to remove mixture; 6-X, VIIIC-Mixture, Exonuclease VIII, truncated within CutSmart buffer to remove mixture; 4-X: I+III-Mixture, I+III to remove mixture. Loading samples for agarose gel electrophoresis were purified by phenol-chloroform.

    Journal: Nucleic Acids Research

    Article Title: Exonuclease combinations reduce noises in 3D genomics technologies

    doi: 10.1093/nar/gkaa106

    Figure Lengend Snippet: Experimental results for exonuclease combinations treatment. ( A ) The cleavage mechanism of Lamada and Exonuclease I combinations. Other exonuclease combinations (Lambda and RecJF; Exonuclease I and Exonuclease III) are shown in Supplementary Figure S1A and Supplementary Table S1 . ( B ) Three exonuclease combinations (LRL; LRC; LIC) removed linear DNA from a paradigm mixture. M, 1 kb DNA ladder; P, pGL4.23 plasmid; L, linearized plasmid; X, mixture (plasmid and linear DNA 1:1); 1-X, LRL-Mixture, LRL to cut mixture; 2-X, LRC-Mixture, LRC to cut mixture; 3-X, LIC-Mixture, LIC to cut mixture; 3-P, LIC to cut plasmid; 3-L, LIC-Lin, LIC to cut linearized plasmid; MS, supercoiled ladder. ( C ) I+III combination and Exonuclease VIII, truncated elimination tests. 5-X, VIII4-Mixture, Exonuclease VIII, truncated within Buffer 4 to remove mixture; 6-X, VIIIC-Mixture, Exonuclease VIII, truncated within CutSmart buffer to remove mixture; 4-X: I+III-Mixture, I+III to remove mixture. Loading samples for agarose gel electrophoresis were purified by phenol-chloroform.

    Article Snippet: After LRL (Lambda and RecJF within Lambda buffer) (NEB), LRC (Lambda and RecJF within Cutsmart buffer) (NEB), LIC (Lambda and Exonuclease I within Cutsmart buffer) (NEB) and I+IIIC (I+III or IIIIC) (Exonuclease I and Exonuclease III within Cutsmart buffer) (NEB) elimination, qPCR was performed.

    Techniques: Plasmid Preparation, Agarose Gel Electrophoresis, Purification

    Analysis of pUC19 DNA following treatment with clb − or clb + E. coli and linearization with the restriction enzyme EcoRI. The cross-linked linearized pUC19 DNA isolated from a co-culture with clb + BW25113 E. coli was used a positive control. A. Analysis of DNA by native gel electrophoresis. B. Analysis of DNA by denaturing gel electrophoresis. For both A and B: DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); linearized pUC19 DNA co-cultured with clb + BW25113 E. coli (Lane #4); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #5), reacted with buffer (Lane #6), reacted with EcoRI restriction enzyme (Lane #7); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #8), reacted with buffer (Lane #9), reacted with EcoRI restriction enzyme (Lane #10). Conditions (Lane #4): linearized pUC19 DNA, clb + BW25113 E. coli , M9-CA media, 4 h at 37 °C. Conditions (Lane #5–#7): circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane #5); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #6); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #7). Conditions (Lane #8–#10): circular pUC19 DNA isolated from co-culture with BW25113 clb + E. coli. in in M9-CA media for 4 h at 37 °C (Lane # 8); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #9); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #10). The DNA was isolated and analyzed by native ( Fig. 5A ) or 0.4% NaOH denaturing ( Fig. 5B ) agarose gel electrophoresis (90 V, 1.5 h).

    Journal: bioRxiv

    Article Title: Depurination of colibactin-derived interstrand cross-links

    doi: 10.1101/869313

    Figure Lengend Snippet: Analysis of pUC19 DNA following treatment with clb − or clb + E. coli and linearization with the restriction enzyme EcoRI. The cross-linked linearized pUC19 DNA isolated from a co-culture with clb + BW25113 E. coli was used a positive control. A. Analysis of DNA by native gel electrophoresis. B. Analysis of DNA by denaturing gel electrophoresis. For both A and B: DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); linearized pUC19 DNA co-cultured with clb + BW25113 E. coli (Lane #4); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #5), reacted with buffer (Lane #6), reacted with EcoRI restriction enzyme (Lane #7); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #8), reacted with buffer (Lane #9), reacted with EcoRI restriction enzyme (Lane #10). Conditions (Lane #4): linearized pUC19 DNA, clb + BW25113 E. coli , M9-CA media, 4 h at 37 °C. Conditions (Lane #5–#7): circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane #5); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #6); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #7). Conditions (Lane #8–#10): circular pUC19 DNA isolated from co-culture with BW25113 clb + E. coli. in in M9-CA media for 4 h at 37 °C (Lane # 8); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #9); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #10). The DNA was isolated and analyzed by native ( Fig. 5A ) or 0.4% NaOH denaturing ( Fig. 5B ) agarose gel electrophoresis (90 V, 1.5 h).

    Article Snippet: The CutSmart® buffer (New England Biolabs®) contains 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, and 100 µg/mL BSA.

    Techniques: Isolation, Co-Culture Assay, Positive Control, Nucleic Acid Electrophoresis, Cell Culture, Agarose Gel Electrophoresis

    Workflow for constructing expression clone containing two target-sgRNA expression cassettes with Golden Gate clone. Primers containing adaptors for Golden Gate cloning (OJH307 and OJH308) were used in the amplification with PJG090 as the template. The reagents were recommended as following: one ul of PCR product, 50 ng of PJG112, one ul of Cutsmart Buffer (NEB), 0.4 ul of T4 ligase buffer (NEB), 5 U of Bsa I (NEB), 20 U of T4 DNA ligase (NEB) and add ddH 2 O to 10 ul. The reaction was incubated for 20–25 cycles (37 °C 2 min, 20 °C 5 min), followed by incubation at 50 °C and 80 °C for 5 min, respectively. Subsequently, one ul of the product was introduced into Trans T1 competent cells. Positive clones were identified by clone PCR and sequenced.

    Journal: PeerJ

    Article Title: A simple and efficient cloning system for CRISPR/Cas9-mediated genome editing in rice

    doi: 10.7717/peerj.8491

    Figure Lengend Snippet: Workflow for constructing expression clone containing two target-sgRNA expression cassettes with Golden Gate clone. Primers containing adaptors for Golden Gate cloning (OJH307 and OJH308) were used in the amplification with PJG090 as the template. The reagents were recommended as following: one ul of PCR product, 50 ng of PJG112, one ul of Cutsmart Buffer (NEB), 0.4 ul of T4 ligase buffer (NEB), 5 U of Bsa I (NEB), 20 U of T4 DNA ligase (NEB) and add ddH 2 O to 10 ul. The reaction was incubated for 20–25 cycles (37 °C 2 min, 20 °C 5 min), followed by incubation at 50 °C and 80 °C for 5 min, respectively. Subsequently, one ul of the product was introduced into Trans T1 competent cells. Positive clones were identified by clone PCR and sequenced.

    Article Snippet: The reagents were recommended as following: one ul of PCR product, 50 ng of PJG112, 1 ul of Cutsmart Buffer (NEB), 0.4 ul of T4 ligase buffer (NEB), 5 U of Bsa I (NEB), 20 U of T4 DNA ligase (NEB) and add ddH2 O to 10 ul.

    Techniques: Expressing, Clone Assay, Amplification, Polymerase Chain Reaction, Incubation