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    CutSmart Buffer
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    CutSmart Buffer 5 0 ml
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    New England Biolabs lrc
    CutSmart Buffer
    CutSmart Buffer 5 0 ml
    https://www.bioz.com/result/lrc/product/New England Biolabs
    Average 99 stars, based on 1247 article reviews
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    lrc - by Bioz Stars, 2020-09
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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

    2) 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

    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

    3) Product Images from "Depurination of colibactin-derived interstrand cross-links"

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

    Journal: bioRxiv

    doi: 10.1101/869313

    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).
    Figure Legend 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).

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

    4) Product Images from "Transcription-induced formation of extrachromosomal DNA during yeast ageing"

    Article Title: Transcription-induced formation of extrachromosomal DNA during yeast ageing

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.3000471

    Mus81 is required for eccDNA formation in old and young cells. (A) Southern blot analysis of CUP1 eccDNA and rDNA-derived ERCs in wild-type, mus81 Δ, yen1 Δ, and slx4 Δ cells aged for 24 hours in the presence or absence of 1 mM CuSO 4 , performed as in Fig 1B . (B) Quantification of CUP1 eccDNA and rDNA-derived ERCs in wild-type and mus81 Δ cells aged for 24 hours in the presence or absence of 1 mM CuSO 4 , performed and analysed as in Fig 1B , n = 4. (C) REC-seq analysis of mus81 Δ cells compared to wild type in the absence (left) or presence (right) of 1 mM CuSO 4 . Experiment and analysis as in Fig 4D . (D) Southern blot analysis of eccDNA from the 17 copy P GAL1 -3HA cup1 tandem repeat in non–age-selected BY4741 haploid cell background lacking MEP modifications. P GAL1 -3HA wild-type, sae2 Δ, and mus81 Δ cells were pregrown on YP Raffinose before a 6 hour induction with 2% galactose or 2% glucose. Genomic DNA was digested with Xho I; then 95% of the sample was further digested with ExoV and ExoI; 5% total DNA (lanes 1–6) and 95% ExoV digested material (lanes 7–12) are shown. These cells contain an additional pRS316- CUP1 plasmid to complement the loss of active chromosomal CUP1 genes, labelled as CUP1 plasmid. This plasmid contains an Xho I site and is hence linearised by Xho I and degraded by ExoV. Signals from same membrane stripped and reprobed for rDNA show ERC species. Abundances of eccDNA and ERCs were compared by one-way ANOVA; n = 4 biological replicates; data were log transformed for testing to fulfil the assumptions of a parametric test. (E) Colony formation assay performed on P GAL1 -3HA wild-type and rad52 Δ cells along with BY4741 wild-type and rad52 Δ controls. Cells were pregrown as above on YP raffinose, then serial dilutions from 10 4 to 10 1 cells spotted on YPD and YPGal plates, which were grown at 30°C until control cells had formed equivalent sized colonies (2–3 days). The data underlying this figure may be found in S1 Data and S1 Raw Images . eccDNA, extrachromosomal circular DNA; ERC, extrachromosomal ribosomal DNA circle; ExoV, exonuclease V; rDNA, ribosomal DNA; REC-seq, restriction-digested extrachromosomal circular DNA sequencing.
    Figure Legend Snippet: Mus81 is required for eccDNA formation in old and young cells. (A) Southern blot analysis of CUP1 eccDNA and rDNA-derived ERCs in wild-type, mus81 Δ, yen1 Δ, and slx4 Δ cells aged for 24 hours in the presence or absence of 1 mM CuSO 4 , performed as in Fig 1B . (B) Quantification of CUP1 eccDNA and rDNA-derived ERCs in wild-type and mus81 Δ cells aged for 24 hours in the presence or absence of 1 mM CuSO 4 , performed and analysed as in Fig 1B , n = 4. (C) REC-seq analysis of mus81 Δ cells compared to wild type in the absence (left) or presence (right) of 1 mM CuSO 4 . Experiment and analysis as in Fig 4D . (D) Southern blot analysis of eccDNA from the 17 copy P GAL1 -3HA cup1 tandem repeat in non–age-selected BY4741 haploid cell background lacking MEP modifications. P GAL1 -3HA wild-type, sae2 Δ, and mus81 Δ cells were pregrown on YP Raffinose before a 6 hour induction with 2% galactose or 2% glucose. Genomic DNA was digested with Xho I; then 95% of the sample was further digested with ExoV and ExoI; 5% total DNA (lanes 1–6) and 95% ExoV digested material (lanes 7–12) are shown. These cells contain an additional pRS316- CUP1 plasmid to complement the loss of active chromosomal CUP1 genes, labelled as CUP1 plasmid. This plasmid contains an Xho I site and is hence linearised by Xho I and degraded by ExoV. Signals from same membrane stripped and reprobed for rDNA show ERC species. Abundances of eccDNA and ERCs were compared by one-way ANOVA; n = 4 biological replicates; data were log transformed for testing to fulfil the assumptions of a parametric test. (E) Colony formation assay performed on P GAL1 -3HA wild-type and rad52 Δ cells along with BY4741 wild-type and rad52 Δ controls. Cells were pregrown as above on YP raffinose, then serial dilutions from 10 4 to 10 1 cells spotted on YPD and YPGal plates, which were grown at 30°C until control cells had formed equivalent sized colonies (2–3 days). The data underlying this figure may be found in S1 Data and S1 Raw Images . eccDNA, extrachromosomal circular DNA; ERC, extrachromosomal ribosomal DNA circle; ExoV, exonuclease V; rDNA, ribosomal DNA; REC-seq, restriction-digested extrachromosomal circular DNA sequencing.

    Techniques Used: Southern Blot, Derivative Assay, Plasmid Preparation, Transformation Assay, Colony Assay, DNA Sequencing

    5) Product Images from "Circular synthesized CRISPR/Cas gRNAs for functional interrogations in the coding and noncoding genome"

    Article Title: Circular synthesized CRISPR/Cas gRNAs for functional interrogations in the coding and noncoding genome

    Journal: eLife

    doi: 10.7554/eLife.42549

    Quality control of the TGW validation library. ( A ) Gel electrophoresis visualizing the quality of P1 and P2 preparations of the TGW validation library (4,232 gRNAs). Please note the absence of the 3-kb DNA fragment in the final (P2) validation library. ( B ) The distributions of the TGW validation library P1 and P2 preparations visualized as Lorenz curves. The pre-I-SceI-digested library (P1) and the post-I-SceI-digested library (P2) have similar gRNA distributions. The low area under the curve (AUC) values of 0.65 (P1) and 0.64 (P2) indicate that the I-SceI clean-up digestion does not affect the distribution of gRNAs in the final product.
    Figure Legend Snippet: Quality control of the TGW validation library. ( A ) Gel electrophoresis visualizing the quality of P1 and P2 preparations of the TGW validation library (4,232 gRNAs). Please note the absence of the 3-kb DNA fragment in the final (P2) validation library. ( B ) The distributions of the TGW validation library P1 and P2 preparations visualized as Lorenz curves. The pre-I-SceI-digested library (P1) and the post-I-SceI-digested library (P2) have similar gRNA distributions. The low area under the curve (AUC) values of 0.65 (P1) and 0.64 (P2) indicate that the I-SceI clean-up digestion does not affect the distribution of gRNAs in the final product.

    Techniques Used: Nucleic Acid Electrophoresis

    Quality control and gRNA distributions of the randomized libraries. ( A ) Gel electrophoresis of P1 3Cs libraries, generated with randomized nucleotide positions (related to Figure 2A ). Template pLentiCRISPRv2 is linearized by I-SceI digests, whereas only P1 libraries are partially I-SceI digested. P2 libraries are unaffected by I-SceI digests, demonstrating their high purity. ( B ) The distribution of the randomized nucleotide libraries, derived from panel (A), visualized with Lorenz curves. The AUC values indicate that 3Cs uncouples sequence distribution from sequence diversity.
    Figure Legend Snippet: Quality control and gRNA distributions of the randomized libraries. ( A ) Gel electrophoresis of P1 3Cs libraries, generated with randomized nucleotide positions (related to Figure 2A ). Template pLentiCRISPRv2 is linearized by I-SceI digests, whereas only P1 libraries are partially I-SceI digested. P2 libraries are unaffected by I-SceI digests, demonstrating their high purity. ( B ) The distribution of the randomized nucleotide libraries, derived from panel (A), visualized with Lorenz curves. The AUC values indicate that 3Cs uncouples sequence distribution from sequence diversity.

    Techniques Used: Nucleic Acid Electrophoresis, Generated, Derivative Assay, Sequencing

    6) Product Images from "ERCC1-XPF Interacts with Topoisomerase IIβ to Facilitate the Repair of Activity-induced DNA Breaks"

    Article Title: ERCC1-XPF Interacts with Topoisomerase IIβ to Facilitate the Repair of Activity-induced DNA Breaks

    Journal: bioRxiv

    doi: 10.1101/2020.01.03.892703

    Genome-wide mapping of DSBs in tRA-treated and Ercc1 −/− MEFs. (A) . Genome-wide enrichment (read count; R.C.) of DNA DSBs (normalized per million mapped reads) on -/+2Kb flanking the TSS in untreated (Untr.), (B) . tRA-treated (tRA) and (C) . Ercc1 −/− MEFs. (D) . BLESS signals quantified by qPCR on the tRA-inducible Cfh , Rarb and Hs3st1 gene promoters, the tRA non-inducible Chordc1 gene promoter, as well as on a non-transcribed intergenic region (-) region in untreated MEFs (Untr.) or MEFs treated with tRA (tRA) or tRA and merbarone (tRA/merb). (E) . Enrichment of ERCC1, TOP IIβ and CTCF proteins on DSB-containing genomic fragments derived from primary MEFs. (F) . Venn’s logic diagrams representing the number of transcription-associated DNA DSBs on XPF-bound genomic sites in untreated (Untr.) and tRA-treated MEFs. (G) . Probability of XPF recruitment on genes by means of log^2RNA*log^2Breaks variable from the RNA-Seq and BLISS data. ( H ). BLESS signals quantified by qPCR on Rarb2 gene promoter in tRA-treated wt. and Ercc1 −/− MEFs. ( I ). BLESS signals quantified by qPCR on the promoters of PrlR and Dio1 genes known to be expressed in the P15 Ercc1 −/− and wt. livers and on the promoter of non-expressed Neun gene. ( J ). BLESS signals quantified by qPCR on the promoter of Dhfr and Prnp genes known to be expressed in the P15 Ercc1 −/− and wt. cerebella and on the promoter of the non-expressed Alb gene. Error bars indicate S.E.M. among n > three biological replicates. Asterisk indicates the significance set at p-value: *≤0.05, **≤0.01, ***≤0.001 (two-tailed Student’s t-test).
    Figure Legend Snippet: Genome-wide mapping of DSBs in tRA-treated and Ercc1 −/− MEFs. (A) . Genome-wide enrichment (read count; R.C.) of DNA DSBs (normalized per million mapped reads) on -/+2Kb flanking the TSS in untreated (Untr.), (B) . tRA-treated (tRA) and (C) . Ercc1 −/− MEFs. (D) . BLESS signals quantified by qPCR on the tRA-inducible Cfh , Rarb and Hs3st1 gene promoters, the tRA non-inducible Chordc1 gene promoter, as well as on a non-transcribed intergenic region (-) region in untreated MEFs (Untr.) or MEFs treated with tRA (tRA) or tRA and merbarone (tRA/merb). (E) . Enrichment of ERCC1, TOP IIβ and CTCF proteins on DSB-containing genomic fragments derived from primary MEFs. (F) . Venn’s logic diagrams representing the number of transcription-associated DNA DSBs on XPF-bound genomic sites in untreated (Untr.) and tRA-treated MEFs. (G) . Probability of XPF recruitment on genes by means of log^2RNA*log^2Breaks variable from the RNA-Seq and BLISS data. ( H ). BLESS signals quantified by qPCR on Rarb2 gene promoter in tRA-treated wt. and Ercc1 −/− MEFs. ( I ). BLESS signals quantified by qPCR on the promoters of PrlR and Dio1 genes known to be expressed in the P15 Ercc1 −/− and wt. livers and on the promoter of non-expressed Neun gene. ( J ). BLESS signals quantified by qPCR on the promoter of Dhfr and Prnp genes known to be expressed in the P15 Ercc1 −/− and wt. cerebella and on the promoter of the non-expressed Alb gene. Error bars indicate S.E.M. among n > three biological replicates. Asterisk indicates the significance set at p-value: *≤0.05, **≤0.01, ***≤0.001 (two-tailed Student’s t-test).

    Techniques Used: Genome Wide, Real-time Polymerase Chain Reaction, Derivative Assay, RNA Sequencing Assay, Two Tailed Test

    7) Product Images from "A simple and efficient cloning system for CRISPR/Cas9-mediated genome editing in rice"

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

    Journal: PeerJ

    doi: 10.7717/peerj.8491

    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.
    Figure Legend 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.

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

    8) Product Images from "Depurination of colibactin-derived interstrand cross-links"

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

    Journal: bioRxiv

    doi: 10.1101/869313

    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).
    Figure Legend 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).

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

    9) 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

    10) Product Images from "Di-copper metallodrugs promote NCI-60 chemotherapy via singlet oxygen and superoxide production with tandem TA/TA and AT/AT oligonucleotide discrimination"

    Article Title: Di-copper metallodrugs promote NCI-60 chemotherapy via singlet oxygen and superoxide production with tandem TA/TA and AT/AT oligonucleotide discrimination

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky105

    ( A ) Mean tail moments of untreated SKOV-3 cells and 1.0 μM Cu-Oda, Cu-Terph and Dox. ( B ) Topoisomerase I unwinding across concentration range 0.10–400 μM for Cu-Oda and Cu-Terph . ( C ). DSBs induced by Cu-Oda and Cu-Terph , detected by immunostaining of γH2AX with MFI presented for di -Cu 2+ complexes and Dox. ( D ) CD spectra of Cu-Oda with stDNA and alternating co-polymers poly[d(A⋅T) 2 ] and poly[d(G⋅C) 2 ] (100 μM) at loading ratios 0.01–0.075.
    Figure Legend Snippet: ( A ) Mean tail moments of untreated SKOV-3 cells and 1.0 μM Cu-Oda, Cu-Terph and Dox. ( B ) Topoisomerase I unwinding across concentration range 0.10–400 μM for Cu-Oda and Cu-Terph . ( C ). DSBs induced by Cu-Oda and Cu-Terph , detected by immunostaining of γH2AX with MFI presented for di -Cu 2+ complexes and Dox. ( D ) CD spectra of Cu-Oda with stDNA and alternating co-polymers poly[d(A⋅T) 2 ] and poly[d(G⋅C) 2 ] (100 μM) at loading ratios 0.01–0.075.

    Techniques Used: Concentration Assay, Immunostaining

    11) Product Images from "Droplet Tn-Seq combines microfluidics with Tn-Seq for identifying complex single-cell phenotypes"

    Article Title: Droplet Tn-Seq combines microfluidics with Tn-Seq for identifying complex single-cell phenotypes

    Journal: Nature Communications

    doi: 10.1038/s41467-019-13719-9

    Schematic overview of droplet Tn-Seq. a A microfluidic device encapsulates single bacterial cells into droplets containing growth medium. Bacteria are allowed to grow within droplets, genomic DNA (gDNA) is isolated at the start of the experiment (t1) and after growth (t2). Importantly, while growth for each transposon mutant takes place in isolation, gDNA is isolated from the pooled population, enabling screening of all mutants simultaneously. b gDNA is then amplified with DNA polymerase phi29, digested with MmeI, an adapter is ligated, a ~180 bp fragment is produced which contains ~16 nucleotides of bacterial gDNA, defining the transposon-insertion location, followed by Illumina sequencing. Reads are demultiplexed based on the barcode in the adapter and a potential second barcode in primer 1, mapped to the genome, and fitness is calculated for each defined region.
    Figure Legend Snippet: Schematic overview of droplet Tn-Seq. a A microfluidic device encapsulates single bacterial cells into droplets containing growth medium. Bacteria are allowed to grow within droplets, genomic DNA (gDNA) is isolated at the start of the experiment (t1) and after growth (t2). Importantly, while growth for each transposon mutant takes place in isolation, gDNA is isolated from the pooled population, enabling screening of all mutants simultaneously. b gDNA is then amplified with DNA polymerase phi29, digested with MmeI, an adapter is ligated, a ~180 bp fragment is produced which contains ~16 nucleotides of bacterial gDNA, defining the transposon-insertion location, followed by Illumina sequencing. Reads are demultiplexed based on the barcode in the adapter and a potential second barcode in primer 1, mapped to the genome, and fitness is calculated for each defined region.

    Techniques Used: Isolation, Mutagenesis, Amplification, Produced, Sequencing

    Unbiased whole-genome amplification of low-quantity genomic DNA. a , b gDNA was prepared by two different methods for transposon sequencing. For the WGA sample, 10 ng of gDNA was amplified first with DNA polymerase phi29 before MmeI digestion and adapter ligation. For the standard sample, 1 μg of gDNA was digested with MmeI, followed by adapter ligation. There is a strong correlation between fitness values obtained from WGA preparation compared with standard Tn-Seq library preparation a , and WGA preparation is highly reproducible b .
    Figure Legend Snippet: Unbiased whole-genome amplification of low-quantity genomic DNA. a , b gDNA was prepared by two different methods for transposon sequencing. For the WGA sample, 10 ng of gDNA was amplified first with DNA polymerase phi29 before MmeI digestion and adapter ligation. For the standard sample, 1 μg of gDNA was digested with MmeI, followed by adapter ligation. There is a strong correlation between fitness values obtained from WGA preparation compared with standard Tn-Seq library preparation a , and WGA preparation is highly reproducible b .

    Techniques Used: Whole Genome Amplification, Sequencing, Amplification, Ligation

    12) Product Images from "Restriction Endonucleases from Invasive Neisseria gonorrhoeae Cause Double-Strand Breaks and Distort Mitosis in Epithelial Cells during Infection"

    Article Title: Restriction Endonucleases from Invasive Neisseria gonorrhoeae Cause Double-Strand Breaks and Distort Mitosis in Epithelial Cells during Infection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0114208

    Lysates of N. gonorrhoeae fragments pECFP-N1 and damage DNA from VK2/E6E7 cells. A. DNA agarose gel showing the digestion of pECFP-N1 plasmid by HindIII (positive control, lane 2), MS11 P+ lysate (lane 3), and MS11 P+ HI lysate (lane 5). Lane 5 shows bacterial MS11 P+ lysate without pECFP-N1 and lane 1 shows uncut circular pECFP-N1. B. PFGE analysis of purified VK2/E6E7 genomic DNA treated for 24 h with: lane 1: PBS (negative control), lane 2: MS11 P+ lysate, lane 3: MS11 P+ HI lysate. Lane 4 shows bacterial MS11 P+ lysate without VK2/E6E7 genomic DNA. C. Graph showing quantification of DNA smears (measured directly underneath and below the band). Shown are smear pixel intensities of cellular DNA alone and cellular DNA exposed to bacterial lysates and HI bacterial lysates. D. PFGE showing genomic DNA subjected to commercial restriction enzymes for 24 h. Lane 1: DNA incubated with CutSmart reaction buffer (negative control). Lane 2: DNA incubated with NgoMIV. Lane 3: DNA incubated with MfeI, Lane 4: DNA incubated with NgoMIV and MfeI Lane 5: DNA incubated with NgoMIV and BamHI/KpnI/MfeI (BKM).
    Figure Legend Snippet: Lysates of N. gonorrhoeae fragments pECFP-N1 and damage DNA from VK2/E6E7 cells. A. DNA agarose gel showing the digestion of pECFP-N1 plasmid by HindIII (positive control, lane 2), MS11 P+ lysate (lane 3), and MS11 P+ HI lysate (lane 5). Lane 5 shows bacterial MS11 P+ lysate without pECFP-N1 and lane 1 shows uncut circular pECFP-N1. B. PFGE analysis of purified VK2/E6E7 genomic DNA treated for 24 h with: lane 1: PBS (negative control), lane 2: MS11 P+ lysate, lane 3: MS11 P+ HI lysate. Lane 4 shows bacterial MS11 P+ lysate without VK2/E6E7 genomic DNA. C. Graph showing quantification of DNA smears (measured directly underneath and below the band). Shown are smear pixel intensities of cellular DNA alone and cellular DNA exposed to bacterial lysates and HI bacterial lysates. D. PFGE showing genomic DNA subjected to commercial restriction enzymes for 24 h. Lane 1: DNA incubated with CutSmart reaction buffer (negative control). Lane 2: DNA incubated with NgoMIV. Lane 3: DNA incubated with MfeI, Lane 4: DNA incubated with NgoMIV and MfeI Lane 5: DNA incubated with NgoMIV and BamHI/KpnI/MfeI (BKM).

    Techniques Used: Agarose Gel Electrophoresis, Plasmid Preparation, Positive Control, Purification, Negative Control, Incubation

    13) Product Images from "Identification of an Intermediate in Hepatitis B Virus Covalently Closed Circular (CCC) DNA Formation and Sensitive and Selective CCC DNA Detection"

    Article Title: Identification of an Intermediate in Hepatitis B Virus Covalently Closed Circular (CCC) DNA Formation and Sensitive and Selective CCC DNA Detection

    Journal: Journal of Virology

    doi: 10.1128/JVI.00539-17

    Exo I III versus Exo T5 digestion of plasmid DNA. (A) Diagrams showing expected digestion results of various plasmid DNA species. A break in the circle denotes the nick on the DNA strand. (B and C) Plasmid pCI-HBc (2.5 ng) was mixed with 20 μl of mock PF DNA extracted from uninduced HepAD38 cells. The DNA mix was first treated with Nb.BbvCI (5 units) to nick the plasmid DNA specifically on the minus strand (B and C, lanes 5 to 8) or was left untreated (B and C, lanes 1 to 4) before digestion with Exo I III (5 units and 25 units, respectively) in two different buffers or with Exo T5 (5 units). The DNA samples were then resolved on an agarose gel, and HBc DNA was detected by Southern blotting using a riboprobe specific for the viral plus-strand (B) or minus-strand (C) DNA. The diagrams on the right of panel C depict the various DNA species and their migration on the gel. B3, 1× NEB buffer 3; BCS, 1× NEB buffer Cutsmart; PE, phenol extraction.
    Figure Legend Snippet: Exo I III versus Exo T5 digestion of plasmid DNA. (A) Diagrams showing expected digestion results of various plasmid DNA species. A break in the circle denotes the nick on the DNA strand. (B and C) Plasmid pCI-HBc (2.5 ng) was mixed with 20 μl of mock PF DNA extracted from uninduced HepAD38 cells. The DNA mix was first treated with Nb.BbvCI (5 units) to nick the plasmid DNA specifically on the minus strand (B and C, lanes 5 to 8) or was left untreated (B and C, lanes 1 to 4) before digestion with Exo I III (5 units and 25 units, respectively) in two different buffers or with Exo T5 (5 units). The DNA samples were then resolved on an agarose gel, and HBc DNA was detected by Southern blotting using a riboprobe specific for the viral plus-strand (B) or minus-strand (C) DNA. The diagrams on the right of panel C depict the various DNA species and their migration on the gel. B3, 1× NEB buffer 3; BCS, 1× NEB buffer Cutsmart; PE, phenol extraction.

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Southern Blot, Migration

    Exo I III versus Exo T5 digestion of HBV core and PF DNA. (A) Diagrams showing expected results of digestion with various HBV PF DNA species. Left, structures of known and potential HBV PF DNA species; middle and right, expected digestion products of the various DNA species. The DNA species in the rectangular box, with a covalently closed minus strand and an open plus strand, represents a potential intermediate during RC DNA to CCC DNA conversion that was identified in the current study (see the text for details). The black dot at the 5′ end of the minus strand of the PF-RC and PF-DSL DNA denotes the unknown modification of this end upon removal of the RT protein (deproteination; see the text for details). (B and C) HBV core DNA (0.3 μl) combined with mock PF DNA (20 μl) extracted from uninduced HepAD38 cells (B) or PF DNA (20 μl) extracted from induced HepAD38 cells (C) was treated with Exo I III (5 units and 25 units, respectively) (lanes 3 and 10) or Exo T5 (5 units) (lanes 6 and 13) in 1× NEB CutSmart buffer. Subsequently, MfeI-HF (10 units) was used to linearize CCC DNA (lanes 5, 7, 12, and 14) and Exo T5 (5 units) was used to digest the SS circular DNA (lanes 4 and 11). Heat treatment (95°C, 10 min) was used to denature RC DNA to SS linear DNA (lanes 2 and 9). The DNA samples were then resolved on an agarose gel, and the various HBV DNA species were detected by Southern blotting using a riboprobe specific for the plus-strand (lanes 1 to 7) or minus-strand (lanes 8 to 14) DNA. The diagrams on the sides depict the various DNA species and their migration on the gel. The positions of the various RC DNA species, CCC DNA species, and SS linear and circular DNA species are indicated by the schematic diagrams. Note that the linearized CCC DNA comigrates with the DSL DNA, a minor form present in both core DNA and PF DNA (lanes 1 and 8).
    Figure Legend Snippet: Exo I III versus Exo T5 digestion of HBV core and PF DNA. (A) Diagrams showing expected results of digestion with various HBV PF DNA species. Left, structures of known and potential HBV PF DNA species; middle and right, expected digestion products of the various DNA species. The DNA species in the rectangular box, with a covalently closed minus strand and an open plus strand, represents a potential intermediate during RC DNA to CCC DNA conversion that was identified in the current study (see the text for details). The black dot at the 5′ end of the minus strand of the PF-RC and PF-DSL DNA denotes the unknown modification of this end upon removal of the RT protein (deproteination; see the text for details). (B and C) HBV core DNA (0.3 μl) combined with mock PF DNA (20 μl) extracted from uninduced HepAD38 cells (B) or PF DNA (20 μl) extracted from induced HepAD38 cells (C) was treated with Exo I III (5 units and 25 units, respectively) (lanes 3 and 10) or Exo T5 (5 units) (lanes 6 and 13) in 1× NEB CutSmart buffer. Subsequently, MfeI-HF (10 units) was used to linearize CCC DNA (lanes 5, 7, 12, and 14) and Exo T5 (5 units) was used to digest the SS circular DNA (lanes 4 and 11). Heat treatment (95°C, 10 min) was used to denature RC DNA to SS linear DNA (lanes 2 and 9). The DNA samples were then resolved on an agarose gel, and the various HBV DNA species were detected by Southern blotting using a riboprobe specific for the plus-strand (lanes 1 to 7) or minus-strand (lanes 8 to 14) DNA. The diagrams on the sides depict the various DNA species and their migration on the gel. The positions of the various RC DNA species, CCC DNA species, and SS linear and circular DNA species are indicated by the schematic diagrams. Note that the linearized CCC DNA comigrates with the DSL DNA, a minor form present in both core DNA and PF DNA (lanes 1 and 8).

    Techniques Used: Countercurrent Chromatography, Modification, Agarose Gel Electrophoresis, Southern Blot, Migration

    Confirmation of the closed circular minus strand in the processed RC DNA by BmgBI or Nt.BbvCI and Exo I III digestion. (A and D) Diagrams showing expected results of digestion performed with various HBV PF DNA species. The short line intersecting the circle denotes the site of BmgBI digestion (A) or Nt.BbvCI nicking (D). The presence of the RNA (short gray line) at the 5′ end of the plus strand in RC DNA prevents BmgBI digestion (panel A; arrow blocked by a short line). The black dot at the 5′ end of the minus strand of the PF-RC DNA denotes the unknown modification of this end upon removal of the RT protein. The DNA species indicated in the rectangular box, with a covalently closed minus strand and an open plus strand, represents a potential intermediate during RC DNA to CCC DNA conversion that was identified in this study (see the text for details). (B and C) HBV core DNA (0.3 μl) combined with mock PF DNA (20 μl) extracted from uninduced HepAD38 cells (lanes 1 to 3) or PF DNA (lanes 4 to 6) extracted from induced HepAD38 cells was treated with BmgBI (5 units) in 1× NEB buffer 3 to linearize all supercoiled and nicked CCC DNA (lanes 2, 3, 5, and 6) or was mock treated (lanes 1 and 4). For lanes 3 and 6, the DNA samples were further digested with Exo I III after BmgBI treatment. The samples were then resolved on an agarose gel, and various HBV DNA species were detected by Southern blotting using a riboprobe specific for the viral plus-strand (B) or minus-strand (C) DNA. The diagrams on the right of panel C depict the various DNA species and their migration on the gel. (E) PF DNA extracted from induced HepAD38 cells was treated with Nt.BbvCI (5 units) in 1× NEB Cutsmart buffer to nick all CCC DNA (lanes 3, 4, 7, and 8) or mock treated (lanes 1 and 5). For lanes 4 and 8, the DNA samples were further digested with Exo I III after Nt.BbvCI treatment. The samples were then resolved on an agarose gel, and various HBV DNA species were detected by Southern blotting using a riboprobe specific for the viral plus-strand (lanes 1 to 4) or minus-strand (lanes 5 to 8) DNA. The diagrams on the right depict the various DNA species and their migration on the gel. Marker, the DNA marker lane. The size of the DNA markers is indicated (in kilobase pairs). The blank spaces between the lanes in panels B, C, and E indicate where other lanes from the same gel that were deemed nonessential for this work were cropped out during the preparation of the figure.
    Figure Legend Snippet: Confirmation of the closed circular minus strand in the processed RC DNA by BmgBI or Nt.BbvCI and Exo I III digestion. (A and D) Diagrams showing expected results of digestion performed with various HBV PF DNA species. The short line intersecting the circle denotes the site of BmgBI digestion (A) or Nt.BbvCI nicking (D). The presence of the RNA (short gray line) at the 5′ end of the plus strand in RC DNA prevents BmgBI digestion (panel A; arrow blocked by a short line). The black dot at the 5′ end of the minus strand of the PF-RC DNA denotes the unknown modification of this end upon removal of the RT protein. The DNA species indicated in the rectangular box, with a covalently closed minus strand and an open plus strand, represents a potential intermediate during RC DNA to CCC DNA conversion that was identified in this study (see the text for details). (B and C) HBV core DNA (0.3 μl) combined with mock PF DNA (20 μl) extracted from uninduced HepAD38 cells (lanes 1 to 3) or PF DNA (lanes 4 to 6) extracted from induced HepAD38 cells was treated with BmgBI (5 units) in 1× NEB buffer 3 to linearize all supercoiled and nicked CCC DNA (lanes 2, 3, 5, and 6) or was mock treated (lanes 1 and 4). For lanes 3 and 6, the DNA samples were further digested with Exo I III after BmgBI treatment. The samples were then resolved on an agarose gel, and various HBV DNA species were detected by Southern blotting using a riboprobe specific for the viral plus-strand (B) or minus-strand (C) DNA. The diagrams on the right of panel C depict the various DNA species and their migration on the gel. (E) PF DNA extracted from induced HepAD38 cells was treated with Nt.BbvCI (5 units) in 1× NEB Cutsmart buffer to nick all CCC DNA (lanes 3, 4, 7, and 8) or mock treated (lanes 1 and 5). For lanes 4 and 8, the DNA samples were further digested with Exo I III after Nt.BbvCI treatment. The samples were then resolved on an agarose gel, and various HBV DNA species were detected by Southern blotting using a riboprobe specific for the viral plus-strand (lanes 1 to 4) or minus-strand (lanes 5 to 8) DNA. The diagrams on the right depict the various DNA species and their migration on the gel. Marker, the DNA marker lane. The size of the DNA markers is indicated (in kilobase pairs). The blank spaces between the lanes in panels B, C, and E indicate where other lanes from the same gel that were deemed nonessential for this work were cropped out during the preparation of the figure.

    Techniques Used: Modification, Countercurrent Chromatography, Agarose Gel Electrophoresis, Southern Blot, Migration, Marker

    14) Product Images from "Di-copper metallodrugs promote NCI-60 chemotherapy via singlet oxygen and superoxide production with tandem TA/TA and AT/AT oligonucleotide discrimination"

    Article Title: Di-copper metallodrugs promote NCI-60 chemotherapy via singlet oxygen and superoxide production with tandem TA/TA and AT/AT oligonucleotide discrimination

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky105

    ( A ) Mean tail moments of untreated SKOV-3 cells and 1.0 μM Cu-Oda, Cu-Terph and Dox. ( B ) Topoisomerase I unwinding across concentration range 0.10–400 μM for Cu-Oda and Cu-Terph . ( C ). DSBs induced by Cu-Oda and Cu-Terph , detected by immunostaining of γH2AX with MFI presented for di -Cu 2+ complexes and Dox. ( D ) CD spectra of Cu-Oda with stDNA and alternating co-polymers poly[d(A⋅T) 2 ] and poly[d(G⋅C) 2 ] (100 μM) at loading ratios 0.01–0.075.
    Figure Legend Snippet: ( A ) Mean tail moments of untreated SKOV-3 cells and 1.0 μM Cu-Oda, Cu-Terph and Dox. ( B ) Topoisomerase I unwinding across concentration range 0.10–400 μM for Cu-Oda and Cu-Terph . ( C ). DSBs induced by Cu-Oda and Cu-Terph , detected by immunostaining of γH2AX with MFI presented for di -Cu 2+ complexes and Dox. ( D ) CD spectra of Cu-Oda with stDNA and alternating co-polymers poly[d(A⋅T) 2 ] and poly[d(G⋅C) 2 ] (100 μM) at loading ratios 0.01–0.075.

    Techniques Used: Concentration Assay, Immunostaining

    15) Product Images from "Depurination of colibactin-derived interstrand cross-links"

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

    Journal: bioRxiv

    doi: 10.1101/869313

    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).
    Figure Legend 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).

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

    16) Product Images from "Identification of an Intermediate in Hepatitis B Virus Covalently Closed Circular (CCC) DNA Formation and Sensitive and Selective CCC DNA Detection"

    Article Title: Identification of an Intermediate in Hepatitis B Virus Covalently Closed Circular (CCC) DNA Formation and Sensitive and Selective CCC DNA Detection

    Journal: Journal of Virology

    doi: 10.1128/JVI.00539-17

    Exo I III versus Exo T5 digestion of plasmid DNA. (A) Diagrams showing expected digestion results of various plasmid DNA species. A break in the circle denotes the nick on the DNA strand. (B and C) Plasmid pCI-HBc (2.5 ng) was mixed with 20 μl of mock PF DNA extracted from uninduced HepAD38 cells. The DNA mix was first treated with Nb.BbvCI (5 units) to nick the plasmid DNA specifically on the minus strand (B and C, lanes 5 to 8) or was left untreated (B and C, lanes 1 to 4) before digestion with Exo I III (5 units and 25 units, respectively) in two different buffers or with Exo T5 (5 units). The DNA samples were then resolved on an agarose gel, and HBc DNA was detected by Southern blotting using a riboprobe specific for the viral plus-strand (B) or minus-strand (C) DNA. The diagrams on the right of panel C depict the various DNA species and their migration on the gel. B3, 1× NEB buffer 3; BCS, 1× NEB buffer Cutsmart; PE, phenol extraction.
    Figure Legend Snippet: Exo I III versus Exo T5 digestion of plasmid DNA. (A) Diagrams showing expected digestion results of various plasmid DNA species. A break in the circle denotes the nick on the DNA strand. (B and C) Plasmid pCI-HBc (2.5 ng) was mixed with 20 μl of mock PF DNA extracted from uninduced HepAD38 cells. The DNA mix was first treated with Nb.BbvCI (5 units) to nick the plasmid DNA specifically on the minus strand (B and C, lanes 5 to 8) or was left untreated (B and C, lanes 1 to 4) before digestion with Exo I III (5 units and 25 units, respectively) in two different buffers or with Exo T5 (5 units). The DNA samples were then resolved on an agarose gel, and HBc DNA was detected by Southern blotting using a riboprobe specific for the viral plus-strand (B) or minus-strand (C) DNA. The diagrams on the right of panel C depict the various DNA species and their migration on the gel. B3, 1× NEB buffer 3; BCS, 1× NEB buffer Cutsmart; PE, phenol extraction.

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Southern Blot, Migration

    Exo I III versus Exo T5 digestion of HBV core and PF DNA. (A) Diagrams showing expected results of digestion with various HBV PF DNA species. Left, structures of known and potential HBV PF DNA species; middle and right, expected digestion products of the various DNA species. The DNA species in the rectangular box, with a covalently closed minus strand and an open plus strand, represents a potential intermediate during RC DNA to CCC DNA conversion that was identified in the current study (see the text for details). The black dot at the 5′ end of the minus strand of the PF-RC and PF-DSL DNA denotes the unknown modification of this end upon removal of the RT protein (deproteination; see the text for details). (B and C) HBV core DNA (0.3 μl) combined with mock PF DNA (20 μl) extracted from uninduced HepAD38 cells (B) or PF DNA (20 μl) extracted from induced HepAD38 cells (C) was treated with Exo I III (5 units and 25 units, respectively) (lanes 3 and 10) or Exo T5 (5 units) (lanes 6 and 13) in 1× NEB CutSmart buffer. Subsequently, MfeI-HF (10 units) was used to linearize CCC DNA (lanes 5, 7, 12, and 14) and Exo T5 (5 units) was used to digest the SS circular DNA (lanes 4 and 11). Heat treatment (95°C, 10 min) was used to denature RC DNA to SS linear DNA (lanes 2 and 9). The DNA samples were then resolved on an agarose gel, and the various HBV DNA species were detected by Southern blotting using a riboprobe specific for the plus-strand (lanes 1 to 7) or minus-strand (lanes 8 to 14) DNA. The diagrams on the sides depict the various DNA species and their migration on the gel. The positions of the various RC DNA species, CCC DNA species, and SS linear and circular DNA species are indicated by the schematic diagrams. Note that the linearized CCC DNA comigrates with the DSL DNA, a minor form present in both core DNA and PF DNA (lanes 1 and 8).
    Figure Legend Snippet: Exo I III versus Exo T5 digestion of HBV core and PF DNA. (A) Diagrams showing expected results of digestion with various HBV PF DNA species. Left, structures of known and potential HBV PF DNA species; middle and right, expected digestion products of the various DNA species. The DNA species in the rectangular box, with a covalently closed minus strand and an open plus strand, represents a potential intermediate during RC DNA to CCC DNA conversion that was identified in the current study (see the text for details). The black dot at the 5′ end of the minus strand of the PF-RC and PF-DSL DNA denotes the unknown modification of this end upon removal of the RT protein (deproteination; see the text for details). (B and C) HBV core DNA (0.3 μl) combined with mock PF DNA (20 μl) extracted from uninduced HepAD38 cells (B) or PF DNA (20 μl) extracted from induced HepAD38 cells (C) was treated with Exo I III (5 units and 25 units, respectively) (lanes 3 and 10) or Exo T5 (5 units) (lanes 6 and 13) in 1× NEB CutSmart buffer. Subsequently, MfeI-HF (10 units) was used to linearize CCC DNA (lanes 5, 7, 12, and 14) and Exo T5 (5 units) was used to digest the SS circular DNA (lanes 4 and 11). Heat treatment (95°C, 10 min) was used to denature RC DNA to SS linear DNA (lanes 2 and 9). The DNA samples were then resolved on an agarose gel, and the various HBV DNA species were detected by Southern blotting using a riboprobe specific for the plus-strand (lanes 1 to 7) or minus-strand (lanes 8 to 14) DNA. The diagrams on the sides depict the various DNA species and their migration on the gel. The positions of the various RC DNA species, CCC DNA species, and SS linear and circular DNA species are indicated by the schematic diagrams. Note that the linearized CCC DNA comigrates with the DSL DNA, a minor form present in both core DNA and PF DNA (lanes 1 and 8).

    Techniques Used: Countercurrent Chromatography, Modification, Agarose Gel Electrophoresis, Southern Blot, Migration

    Confirmation of the closed circular minus strand in the processed RC DNA by BmgBI or Nt.BbvCI and Exo I III digestion. (A and D) Diagrams showing expected results of digestion performed with various HBV PF DNA species. The short line intersecting the circle denotes the site of BmgBI digestion (A) or Nt.BbvCI nicking (D). The presence of the RNA (short gray line) at the 5′ end of the plus strand in RC DNA prevents BmgBI digestion (panel A; arrow blocked by a short line). The black dot at the 5′ end of the minus strand of the PF-RC DNA denotes the unknown modification of this end upon removal of the RT protein. The DNA species indicated in the rectangular box, with a covalently closed minus strand and an open plus strand, represents a potential intermediate during RC DNA to CCC DNA conversion that was identified in this study (see the text for details). (B and C) HBV core DNA (0.3 μl) combined with mock PF DNA (20 μl) extracted from uninduced HepAD38 cells (lanes 1 to 3) or PF DNA (lanes 4 to 6) extracted from induced HepAD38 cells was treated with BmgBI (5 units) in 1× NEB buffer 3 to linearize all supercoiled and nicked CCC DNA (lanes 2, 3, 5, and 6) or was mock treated (lanes 1 and 4). For lanes 3 and 6, the DNA samples were further digested with Exo I III after BmgBI treatment. The samples were then resolved on an agarose gel, and various HBV DNA species were detected by Southern blotting using a riboprobe specific for the viral plus-strand (B) or minus-strand (C) DNA. The diagrams on the right of panel C depict the various DNA species and their migration on the gel. (E) PF DNA extracted from induced HepAD38 cells was treated with Nt.BbvCI (5 units) in 1× NEB Cutsmart buffer to nick all CCC DNA (lanes 3, 4, 7, and 8) or mock treated (lanes 1 and 5). For lanes 4 and 8, the DNA samples were further digested with Exo I III after Nt.BbvCI treatment. The samples were then resolved on an agarose gel, and various HBV DNA species were detected by Southern blotting using a riboprobe specific for the viral plus-strand (lanes 1 to 4) or minus-strand (lanes 5 to 8) DNA. The diagrams on the right depict the various DNA species and their migration on the gel. Marker, the DNA marker lane. The size of the DNA markers is indicated (in kilobase pairs). The blank spaces between the lanes in panels B, C, and E indicate where other lanes from the same gel that were deemed nonessential for this work were cropped out during the preparation of the figure.
    Figure Legend Snippet: Confirmation of the closed circular minus strand in the processed RC DNA by BmgBI or Nt.BbvCI and Exo I III digestion. (A and D) Diagrams showing expected results of digestion performed with various HBV PF DNA species. The short line intersecting the circle denotes the site of BmgBI digestion (A) or Nt.BbvCI nicking (D). The presence of the RNA (short gray line) at the 5′ end of the plus strand in RC DNA prevents BmgBI digestion (panel A; arrow blocked by a short line). The black dot at the 5′ end of the minus strand of the PF-RC DNA denotes the unknown modification of this end upon removal of the RT protein. The DNA species indicated in the rectangular box, with a covalently closed minus strand and an open plus strand, represents a potential intermediate during RC DNA to CCC DNA conversion that was identified in this study (see the text for details). (B and C) HBV core DNA (0.3 μl) combined with mock PF DNA (20 μl) extracted from uninduced HepAD38 cells (lanes 1 to 3) or PF DNA (lanes 4 to 6) extracted from induced HepAD38 cells was treated with BmgBI (5 units) in 1× NEB buffer 3 to linearize all supercoiled and nicked CCC DNA (lanes 2, 3, 5, and 6) or was mock treated (lanes 1 and 4). For lanes 3 and 6, the DNA samples were further digested with Exo I III after BmgBI treatment. The samples were then resolved on an agarose gel, and various HBV DNA species were detected by Southern blotting using a riboprobe specific for the viral plus-strand (B) or minus-strand (C) DNA. The diagrams on the right of panel C depict the various DNA species and their migration on the gel. (E) PF DNA extracted from induced HepAD38 cells was treated with Nt.BbvCI (5 units) in 1× NEB Cutsmart buffer to nick all CCC DNA (lanes 3, 4, 7, and 8) or mock treated (lanes 1 and 5). For lanes 4 and 8, the DNA samples were further digested with Exo I III after Nt.BbvCI treatment. The samples were then resolved on an agarose gel, and various HBV DNA species were detected by Southern blotting using a riboprobe specific for the viral plus-strand (lanes 1 to 4) or minus-strand (lanes 5 to 8) DNA. The diagrams on the right depict the various DNA species and their migration on the gel. Marker, the DNA marker lane. The size of the DNA markers is indicated (in kilobase pairs). The blank spaces between the lanes in panels B, C, and E indicate where other lanes from the same gel that were deemed nonessential for this work were cropped out during the preparation of the figure.

    Techniques Used: Modification, Countercurrent Chromatography, Agarose Gel Electrophoresis, Southern Blot, Migration, Marker

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    Amplification:

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    Magnetic Beads:

    Article Title: Droplet Tn-Seq combines microfluidics with Tn-Seq for identifying complex single-cell phenotypes
    Article Snippet: .. 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 , , .

    Isolation:

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    Article Snippet: .. 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.

    Purification:

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    Real-time Polymerase Chain Reaction:

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    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. .. Primer BH2 for qPCR was designed across both ends of the BamHI restriction site.

    Incubation:

    Article Title: Droplet Tn-Seq combines microfluidics with Tn-Seq for identifying complex single-cell phenotypes
    Article Snippet: .. 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:

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    Plasmid Preparation:

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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: <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.
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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