ecori hf  (New England Biolabs)


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    EcoRI HF
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    EcoRI HF 50 000 units
    Catalog Number:
    R3101L
    Price:
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    Category:
    Restriction Enzymes
    Size:
    50 000 units
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    New England Biolabs ecori hf
    EcoRI HF
    EcoRI HF 50 000 units
    https://www.bioz.com/result/ecori hf/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Images

    1) Product Images from "Genetic transformation of Spizellomyces punctatus, a resource for studying chytrid biology and evolutionary cell biology"

    Article Title: Genetic transformation of Spizellomyces punctatus, a resource for studying chytrid biology and evolutionary cell biology

    Journal: eLife

    doi: 10.7554/eLife.52741

    Mapping T-DNA genomic insertion sites with inverse PCR. ( A ) Diagram of T-DNA integration and the location of PCR/sequencing primers and restriction sites used for inverse PCR (invPCR). We only show primers adjacent to the left border (LB) because they consistently amplified for all transformants, unlike the primers adjacent to the right border. ( B ) Example of amplification by invPCR of the LB-genome border after EcoRI genomic digestion and ligation for an untransformed strain (WT), four independent transformants and non-template control. ( C ) Amplification by invPCR of the LB-genome border after HindIII genomic digestion and ligation. T-DNA location for all transformants was confirmed by two independent biological replicates (i.e. independent genomic extractions, ligation and invPCR). ( D ) T-DNA insertion sites in four independent transformants of Spizellomyces . In strain EM20C-3, invPCR for EcoRI indicated LB is located toward SPPG_02523, while invPCR for HindIII shows same insertion site but with an inverted direction. The divergent invPCR results might represent an insertion of a tandem inverted T-DNA. ( E ) Three of the four strains (EM20C-2,3,4) have similar tdTomato fluorescence levels as determined by flow cytometry.
    Figure Legend Snippet: Mapping T-DNA genomic insertion sites with inverse PCR. ( A ) Diagram of T-DNA integration and the location of PCR/sequencing primers and restriction sites used for inverse PCR (invPCR). We only show primers adjacent to the left border (LB) because they consistently amplified for all transformants, unlike the primers adjacent to the right border. ( B ) Example of amplification by invPCR of the LB-genome border after EcoRI genomic digestion and ligation for an untransformed strain (WT), four independent transformants and non-template control. ( C ) Amplification by invPCR of the LB-genome border after HindIII genomic digestion and ligation. T-DNA location for all transformants was confirmed by two independent biological replicates (i.e. independent genomic extractions, ligation and invPCR). ( D ) T-DNA insertion sites in four independent transformants of Spizellomyces . In strain EM20C-3, invPCR for EcoRI indicated LB is located toward SPPG_02523, while invPCR for HindIII shows same insertion site but with an inverted direction. The divergent invPCR results might represent an insertion of a tandem inverted T-DNA. ( E ) Three of the four strains (EM20C-2,3,4) have similar tdTomato fluorescence levels as determined by flow cytometry.

    Techniques Used: Inverse PCR, Polymerase Chain Reaction, Sequencing, Amplification, Ligation, Fluorescence, Flow Cytometry

    2) Product Images from "DNA translocase repositions a nucleosome by the lane-switch mechanism"

    Article Title: DNA translocase repositions a nucleosome by the lane-switch mechanism

    Journal: bioRxiv

    doi: 10.1101/2021.02.15.431322

    Restriction enzyme digestion assay. (A) The DNA sequence map of substrates used in the assay. The T7 RNAP stall sites where the translocase first encounter adenine are marked by the black arrows. BssSI and EcoRI restriction sites are marked by the blue and red arrows, respectively. (B) Cartoons of the experimental setup. At the pre-transcription stage, the BssSI site, but not the EcoRI site, is occluded by the histone core complex. On the other hand, if the histone core complex repositions upon transcription according to the lane-switch mechanism, the EcoRI site, but not the BssSI site, is occluded by the histone core complex. (C) Images of 1% agarose gel on which the EcoRI-digested products run. The DNA substrates were digested at the pre- and post-transcription stages. (D) Plot showing the intensity of the undigested band at pre-transcription stage divided by that of the post-transcription stage (Δ occlusion ). The assay was repeated using naked DNA (Naked), nucleosome reconstituted DNA (Nucleosome), and the naked DNA with histone core complexes in solution (Octamer).
    Figure Legend Snippet: Restriction enzyme digestion assay. (A) The DNA sequence map of substrates used in the assay. The T7 RNAP stall sites where the translocase first encounter adenine are marked by the black arrows. BssSI and EcoRI restriction sites are marked by the blue and red arrows, respectively. (B) Cartoons of the experimental setup. At the pre-transcription stage, the BssSI site, but not the EcoRI site, is occluded by the histone core complex. On the other hand, if the histone core complex repositions upon transcription according to the lane-switch mechanism, the EcoRI site, but not the BssSI site, is occluded by the histone core complex. (C) Images of 1% agarose gel on which the EcoRI-digested products run. The DNA substrates were digested at the pre- and post-transcription stages. (D) Plot showing the intensity of the undigested band at pre-transcription stage divided by that of the post-transcription stage (Δ occlusion ). The assay was repeated using naked DNA (Naked), nucleosome reconstituted DNA (Nucleosome), and the naked DNA with histone core complexes in solution (Octamer).

    Techniques Used: Sequencing, Agarose Gel Electrophoresis

    The images of 1% agarose gel on which the digested products run. The DNA substrates were digested by BssSI and EcoRI at the pre-and post-transcription stages. Depending on the substrates, the RNAP stalled at −54 bps (top), −29 bps (middle), and −14 bps (bottom), respectively. The assay was repeated using naked DNA (Naked), nucleosome reconstituted DNA (Nucleosome), and the naked DNA with histone core complexes in solution (Octamer). The marker is Quick-Load Purple 50 bp DNA Ladder (New England BioLabs; N0556S).
    Figure Legend Snippet: The images of 1% agarose gel on which the digested products run. The DNA substrates were digested by BssSI and EcoRI at the pre-and post-transcription stages. Depending on the substrates, the RNAP stalled at −54 bps (top), −29 bps (middle), and −14 bps (bottom), respectively. The assay was repeated using naked DNA (Naked), nucleosome reconstituted DNA (Nucleosome), and the naked DNA with histone core complexes in solution (Octamer). The marker is Quick-Load Purple 50 bp DNA Ladder (New England BioLabs; N0556S).

    Techniques Used: Agarose Gel Electrophoresis, Marker

    3) Product Images from "The Chd1 chromatin remodeler can sense both entry and exit sides of the nucleosome"

    Article Title: The Chd1 chromatin remodeler can sense both entry and exit sides of the nucleosome

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw406

    Exit side DNA defines the extent that Chd1 slides nucleosomes in the presence of Lac repressor. ( A ) Nucleosomes are dynamically shifted back-and-forth by Chd1 in the presence of LacI. End-positioned 0N70 nucleosomes containing a LacO(−11) site on the 70 bp side and an EcoRI cut site just inside the 0 bp side were digested by EcoRI and monitored by SDS-PAGE. The slower rate of digestion in the absence of ATP (gray) demonstrates that the EcoRI site is initially buried. In the presence of Chd1 and ATP, the same fraction of nucleosomal DNA becomes cleaved in the presence (filled circle) or absence (open circle) of LacI, demonstrating dynamic repositioning by Chd1 in the presence of LacI. Based on single exponential fits to the data, the sliding rate in the presence of LacI was calculated to be 7-fold slower than in the absence of LacI. Error bars indicate the standard deviations from five or more independent experiments. ( B ) Comparison of nucleosome sliding reactions carried out in the absence and presence of LacI, using (−10)N80[LacO-11R] substrates. Time points for these experiments were 0, 1, 4, 16, 64 min. ( C ) Comparison of the preferred distributions of nucleosome positions for 0N80 and (−10)N80 nucleosomes when Chd1 sliding was carried out in the presence of LacI. White peaks show zero time points and gray peaks are the nucleosome positions at 64 min time points.
    Figure Legend Snippet: Exit side DNA defines the extent that Chd1 slides nucleosomes in the presence of Lac repressor. ( A ) Nucleosomes are dynamically shifted back-and-forth by Chd1 in the presence of LacI. End-positioned 0N70 nucleosomes containing a LacO(−11) site on the 70 bp side and an EcoRI cut site just inside the 0 bp side were digested by EcoRI and monitored by SDS-PAGE. The slower rate of digestion in the absence of ATP (gray) demonstrates that the EcoRI site is initially buried. In the presence of Chd1 and ATP, the same fraction of nucleosomal DNA becomes cleaved in the presence (filled circle) or absence (open circle) of LacI, demonstrating dynamic repositioning by Chd1 in the presence of LacI. Based on single exponential fits to the data, the sliding rate in the presence of LacI was calculated to be 7-fold slower than in the absence of LacI. Error bars indicate the standard deviations from five or more independent experiments. ( B ) Comparison of nucleosome sliding reactions carried out in the absence and presence of LacI, using (−10)N80[LacO-11R] substrates. Time points for these experiments were 0, 1, 4, 16, 64 min. ( C ) Comparison of the preferred distributions of nucleosome positions for 0N80 and (−10)N80 nucleosomes when Chd1 sliding was carried out in the presence of LacI. White peaks show zero time points and gray peaks are the nucleosome positions at 64 min time points.

    Techniques Used: SDS Page

    4) 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

    5) Product Images from "Multiple Pairwise Analysis of Non-homologous Centromere Coupling Reveals Preferential Chromosome Size-Dependent Interactions and a Role for Bouquet Formation in Establishing the Interaction Pattern"

    Article Title: Multiple Pairwise Analysis of Non-homologous Centromere Coupling Reveals Preferential Chromosome Size-Dependent Interactions and a Role for Bouquet Formation in Establishing the Interaction Pattern

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1006347

    3C2D-qPCR design for characterizing centromere coupling. (A) Design of two primers (arrow) and one Taqman probe (ball-and-stick) to quantify the interaction between restriction fragments ligated together, each encompassing a non-homologous centromere (oval). (B) Distribution of restriction enzyme sites on fragments encompassing the centromere ( CEN ) on all 16 chromosomes using an EcoRI single digestion (3C) (left) or an EcoRI-MfeI double digestion (3C2D) (right). For each chromosome (on y-axis), the distances of the restriction sites delimitating the CEN fragment are given in kilobases (kb), in relation to the center of the CEN (x-axis). Blue vertical lines indicate EcoRI sites and red lines indicate MfeI sites.
    Figure Legend Snippet: 3C2D-qPCR design for characterizing centromere coupling. (A) Design of two primers (arrow) and one Taqman probe (ball-and-stick) to quantify the interaction between restriction fragments ligated together, each encompassing a non-homologous centromere (oval). (B) Distribution of restriction enzyme sites on fragments encompassing the centromere ( CEN ) on all 16 chromosomes using an EcoRI single digestion (3C) (left) or an EcoRI-MfeI double digestion (3C2D) (right). For each chromosome (on y-axis), the distances of the restriction sites delimitating the CEN fragment are given in kilobases (kb), in relation to the center of the CEN (x-axis). Blue vertical lines indicate EcoRI sites and red lines indicate MfeI sites.

    Techniques Used: Real-time Polymerase Chain Reaction

    6) Product Images from "Engineered viral DNA polymerase with enhanced DNA amplification capacity: a proof-of-concept of isothermal amplification of damaged DNA"

    Article Title: Engineered viral DNA polymerase with enhanced DNA amplification capacity: a proof-of-concept of isothermal amplification of damaged DNA

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-71773-6

    Isothermal multiple displacement amplification in different conditions. Multiple displacement amplification of plasmid DNA with different GC content ( A ) and in the presence of high salt concentrations ( B ). Plasmid full-length size after EcoRI digestion is indicated with blue or red arrow for pUC19 and pIJ702, respectively.
    Figure Legend Snippet: Isothermal multiple displacement amplification in different conditions. Multiple displacement amplification of plasmid DNA with different GC content ( A ) and in the presence of high salt concentrations ( B ). Plasmid full-length size after EcoRI digestion is indicated with blue or red arrow for pUC19 and pIJ702, respectively.

    Techniques Used: Multiple Displacement Amplification, Plasmid Preparation

    7) Product Images from "Efficient mouse genome engineering by CRISPR-EZ (CRISPR RNP Electroporation of Zygotes) technology"

    Article Title: Efficient mouse genome engineering by CRISPR-EZ (CRISPR RNP Electroporation of Zygotes) technology

    Journal: Nature protocols

    doi: 10.1038/nprot.2018.012

    Optimization of CRISPR-EZ conditions for editing efficiency and embryo viability. (a) A diagram illustrates the NHEJ and HDR editing strategies for exon 1 of the Tyr gene. A successful NHEJ editing ablates a HinfI site and disrupts T yr gene function. A successful HDR editing replaces the HinfI site with an EcoRI site, introducing a frameshift mutation that abolishes Tyr gene function. (b) Representative RFLP results of Tyr edited mice indicate successful NHEJ editing (top) and HDR editing (bottom). (c) Since bi-allelic Tyr deficiency causes albinism in edited mice, the extent of albinism correlates the extent of Tyr editing that disrupts the genes function. Coat color (left) and viability (right) of C57B/6J edited mice generated from 2, 4, 6 or 8 pulse CRISPR-EZ conditions. Viability is defined as the percentage of live animals born out of total embryos transferred. The 6-pulse condition maximizes editing efficiency while minimally impacting pup viability. (d) Comparison of editing efficiency between C57B/6J and C57B/6N mouse strain using 2 or 6-pulse electroporation conditions. The 6-pulse CRISPR-EZ condition is equally effective in both strains. (e-i) Representative images are shown for the coat color of edited mice from experiments shown in (b-d). All animal procedures were approved by the Institutional Animal Care and Use Committee of UC Davis.
    Figure Legend Snippet: Optimization of CRISPR-EZ conditions for editing efficiency and embryo viability. (a) A diagram illustrates the NHEJ and HDR editing strategies for exon 1 of the Tyr gene. A successful NHEJ editing ablates a HinfI site and disrupts T yr gene function. A successful HDR editing replaces the HinfI site with an EcoRI site, introducing a frameshift mutation that abolishes Tyr gene function. (b) Representative RFLP results of Tyr edited mice indicate successful NHEJ editing (top) and HDR editing (bottom). (c) Since bi-allelic Tyr deficiency causes albinism in edited mice, the extent of albinism correlates the extent of Tyr editing that disrupts the genes function. Coat color (left) and viability (right) of C57B/6J edited mice generated from 2, 4, 6 or 8 pulse CRISPR-EZ conditions. Viability is defined as the percentage of live animals born out of total embryos transferred. The 6-pulse condition maximizes editing efficiency while minimally impacting pup viability. (d) Comparison of editing efficiency between C57B/6J and C57B/6N mouse strain using 2 or 6-pulse electroporation conditions. The 6-pulse CRISPR-EZ condition is equally effective in both strains. (e-i) Representative images are shown for the coat color of edited mice from experiments shown in (b-d). All animal procedures were approved by the Institutional Animal Care and Use Committee of UC Davis.

    Techniques Used: CRISPR, Non-Homologous End Joining, Mutagenesis, Mouse Assay, Generated, Electroporation

    8) Product Images from "An exogenous chloroplast genome for complex sequence manipulation in algae"

    Article Title: An exogenous chloroplast genome for complex sequence manipulation in algae

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr1008

    Characterization of the cloned C. reinhardtii chloroplast genome in vivo . ( A ) A nested set representing the presence of increasing numbers of markers in primary transformants of pCr03 into a psbD knockout strain as determined by PCR ( Table 2 ; primers used as follows: M1, 11 606 and 11 607; M2, 5512 and 5513; M3, 11 456 and 11 457; and M4, 14 067 and 14 068.). The broken circle shows the subset of transformants with M1, M2, M3 and M4 that gave rise to the same genotype upon rescreening. ( B–E ) Southern blot analysis of EcoRI (B, C and E) or NdeI (D) digests (see ‘Materials and Methods’ section). Probes were specific for sequences adjacent to integration sites for M1 (B), M2 (C), M3 (D) and M4 (E). All samples are arranged as follows: Lane L, 1 kb DNA ladder (Invitrogen; Carlsbad, CA); lane 1, wild-type; lane 2, purified pCr03; and lane 3, a representative algae clone containing all unique markers. A single band in lane 3 indicates homoplasmic integration of the marker, while two bands indicate heteroplasmy with the wild-type locus.
    Figure Legend Snippet: Characterization of the cloned C. reinhardtii chloroplast genome in vivo . ( A ) A nested set representing the presence of increasing numbers of markers in primary transformants of pCr03 into a psbD knockout strain as determined by PCR ( Table 2 ; primers used as follows: M1, 11 606 and 11 607; M2, 5512 and 5513; M3, 11 456 and 11 457; and M4, 14 067 and 14 068.). The broken circle shows the subset of transformants with M1, M2, M3 and M4 that gave rise to the same genotype upon rescreening. ( B–E ) Southern blot analysis of EcoRI (B, C and E) or NdeI (D) digests (see ‘Materials and Methods’ section). Probes were specific for sequences adjacent to integration sites for M1 (B), M2 (C), M3 (D) and M4 (E). All samples are arranged as follows: Lane L, 1 kb DNA ladder (Invitrogen; Carlsbad, CA); lane 1, wild-type; lane 2, purified pCr03; and lane 3, a representative algae clone containing all unique markers. A single band in lane 3 indicates homoplasmic integration of the marker, while two bands indicate heteroplasmy with the wild-type locus.

    Techniques Used: Clone Assay, In Vivo, Knock-Out, Polymerase Chain Reaction, Southern Blot, Purification, Marker

    9) Product Images from "Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly"

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0189892

    Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with PstI and EcoRI resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.
    Figure Legend Snippet: Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with PstI and EcoRI resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.

    Techniques Used: Construct, Plasmid Preparation, Derivative Assay, Transformation Assay, Nucleic Acid Electrophoresis, Isolation

    10) Product Images from "Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly"

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0189892

    Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with PstI and EcoRI resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.
    Figure Legend Snippet: Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with PstI and EcoRI resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.

    Techniques Used: Construct, Plasmid Preparation, Derivative Assay, Transformation Assay, Nucleic Acid Electrophoresis, Isolation

    11) Product Images from "PCNA-K164 ubiquitination facilitates origin licensing and mitotic DNA synthesis"

    Article Title: PCNA-K164 ubiquitination facilitates origin licensing and mitotic DNA synthesis

    Journal: bioRxiv

    doi: 10.1101/2020.06.25.172361

    Generation of a PCNA K164R mutant cell line in RPE-1 using CRISPR/Cas9 A) Schematic of the human PCNA indicating that exon 5 was targeted by CRISPR-Cas9. The K164R mutation was knocked-in utilizing a donor plasmid. B) Schematic of screening PCR and expected PCR product sizes after EcoRI restriction enzyme digestion. C) Representative genotyping PCR. Not targeted (wildtype; 1426 bp), monoallelic knock-in (KIN) ( PCNA KR/- 1E4; 1426 bp, 1168 bp, 258 bp), and biallelic KIN ( PCNA KR/KR 1E12, 2B10; 1168bp, 258 bp). D) Karyotyping analysis from RPE-1 wildtype, PCNA KR/KR (1E12, 2B10) and PCNA KR/- (1E4) cell lines. Blue indicates expec ted RPE-1 karyotype. Red indicates chromosomal abnormalities. E) Western blot analyses of whole cell extracts from wildtype RPE-1, PCNA K164R , and RAD18 -/- cells for MCM2 with α-Tubulin as the loading control. Quantification of MCM2 levels normalized to loading control.
    Figure Legend Snippet: Generation of a PCNA K164R mutant cell line in RPE-1 using CRISPR/Cas9 A) Schematic of the human PCNA indicating that exon 5 was targeted by CRISPR-Cas9. The K164R mutation was knocked-in utilizing a donor plasmid. B) Schematic of screening PCR and expected PCR product sizes after EcoRI restriction enzyme digestion. C) Representative genotyping PCR. Not targeted (wildtype; 1426 bp), monoallelic knock-in (KIN) ( PCNA KR/- 1E4; 1426 bp, 1168 bp, 258 bp), and biallelic KIN ( PCNA KR/KR 1E12, 2B10; 1168bp, 258 bp). D) Karyotyping analysis from RPE-1 wildtype, PCNA KR/KR (1E12, 2B10) and PCNA KR/- (1E4) cell lines. Blue indicates expec ted RPE-1 karyotype. Red indicates chromosomal abnormalities. E) Western blot analyses of whole cell extracts from wildtype RPE-1, PCNA K164R , and RAD18 -/- cells for MCM2 with α-Tubulin as the loading control. Quantification of MCM2 levels normalized to loading control.

    Techniques Used: Mutagenesis, CRISPR, Plasmid Preparation, Polymerase Chain Reaction, Knock-In, Western Blot

    12) 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

    13) Product Images from "Efficient dual-negative selection for bacterial genome editing"

    Article Title: Efficient dual-negative selection for bacterial genome editing

    Journal: BMC Microbiology

    doi: 10.1186/s12866-020-01819-2

    An optimized method for genome editing in Salmonella enterica . a Map of suicide plasmid pFOK ( aphA , aminoglycoside phosphotransferase gene conferring resistance to kanamycin ; I-sceI gene encoding meganuclease; oriT , origin of conjugational transfer; P tetA , tetA promoter; R6K γ ori , pi-dependent origin of replication; sacB , levansucrase gene; tetR , tetracycline repressor gene; traJ , transcriptional activator for conjugational transfer genes; MCR, multi cloning region containing EcoRI and BamHI recognition sites). b Mechanisms of negative selection for SacB and I-SceI, c Efficiency of negative selection for various chromosomal loci ( sitABCD deletion - orange, foxA deletion - yellow, ssrB point mutation – green, and phoQ chimeric insertion - magenta [ 30 ]) using either SacB or I-SceI, or a combination of both. Fifty colonies were screened for each mutation. d Schematic representation of the consecutive single crossover procedure. Recombination can occur in one of the two homologous sequences (routes 1 and 2). Only alternate single crossover events involving both homologous sequences lead to the desired mutation, while two consecutive single crossovers in the same regions lead to reversion to wild-type (WT) e Overview of the entire procedure. Ideally, each step can be completed in one working day. f Schematic representation of preferential recombination in the right flanking region. External primers 1 and 2 together with plasmid-specific primers oOPC-614 and oOPC-615 can be used to screen co-integrant clones to reveal such bias and to identify rare variants for promoting mutant generation in the second single crossover. g Recombination bias for foxA deletion. PCR results of ex-conjugant screening using external primer 1 (oOPC-396) / oOPC-614 or external primer 2 (oOPC-397) / oOPC-615. Rare ex-conjugants (clones 5, 10) with recombination in the non-preferred flanking region were used for subsequent counter-selection
    Figure Legend Snippet: An optimized method for genome editing in Salmonella enterica . a Map of suicide plasmid pFOK ( aphA , aminoglycoside phosphotransferase gene conferring resistance to kanamycin ; I-sceI gene encoding meganuclease; oriT , origin of conjugational transfer; P tetA , tetA promoter; R6K γ ori , pi-dependent origin of replication; sacB , levansucrase gene; tetR , tetracycline repressor gene; traJ , transcriptional activator for conjugational transfer genes; MCR, multi cloning region containing EcoRI and BamHI recognition sites). b Mechanisms of negative selection for SacB and I-SceI, c Efficiency of negative selection for various chromosomal loci ( sitABCD deletion - orange, foxA deletion - yellow, ssrB point mutation – green, and phoQ chimeric insertion - magenta [ 30 ]) using either SacB or I-SceI, or a combination of both. Fifty colonies were screened for each mutation. d Schematic representation of the consecutive single crossover procedure. Recombination can occur in one of the two homologous sequences (routes 1 and 2). Only alternate single crossover events involving both homologous sequences lead to the desired mutation, while two consecutive single crossovers in the same regions lead to reversion to wild-type (WT) e Overview of the entire procedure. Ideally, each step can be completed in one working day. f Schematic representation of preferential recombination in the right flanking region. External primers 1 and 2 together with plasmid-specific primers oOPC-614 and oOPC-615 can be used to screen co-integrant clones to reveal such bias and to identify rare variants for promoting mutant generation in the second single crossover. g Recombination bias for foxA deletion. PCR results of ex-conjugant screening using external primer 1 (oOPC-396) / oOPC-614 or external primer 2 (oOPC-397) / oOPC-615. Rare ex-conjugants (clones 5, 10) with recombination in the non-preferred flanking region were used for subsequent counter-selection

    Techniques Used: Plasmid Preparation, Clone Assay, Selection, Mutagenesis, Polymerase Chain Reaction

    14) Product Images from "Multiple Phenotypes Resulting from a Mutagenesis Screen for Pharynx Muscle Mutations in Caenorhabditis elegans"

    Article Title: Multiple Phenotypes Resulting from a Mutagenesis Screen for Pharynx Muscle Mutations in Caenorhabditis elegans

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0026594

    PAS136 mapping and pharynx markers. (A) Probable location of the PAS136 pharynx phenotype allele is between 6 cM and 8 cM on LG.I relative to the genetic center of the chromosome (green circle) derived by mapping with DraI or EcoRI specific SNPs corresponding to DNA clones D1007, K02B12, B0205, and F58D5 (orange lines) and between 4.64 cM and 9.2 cM (red circle) using complementation with the deficiency strains MT2179, DC1079, KR2838 and SL536 with overlapping chromosomal deletions (blue lines). (B) pha-4 RNAi used a positive control for pharynx phenotypes, arrow shows lack of myo-2::GFP in most of the head. (C) lam-3 (T22A3.8) RNAi showing a phenotype similar to PAS136 with non-adherent cells (arrow). (D) blmp-1 (F25D7.3) RNAi has a less severe PAS136 phenotype (arrow denotes cell disconnected from the pharynx). (E) hmr-1 (W02B9.1) RNAi results in a Pun phenotype with diminished anterior pharynx cells (arrow). (F) Wild-type MH27 AJM-1 adherens junction antibody staining showing pharynx (ph) and intestine (it) localization. (G) PAS136 embryo with weak and disconnect AJM-1 staining in the pharynx (ph) and more normal AJM-1 in the intestine (it). (H) Wild-type Intermediate Filaments showing three sets of marginal cells (arrows). (I) PAS136 embryo with three sets of marginal cells (arrows). Bar is ∼10 µM.
    Figure Legend Snippet: PAS136 mapping and pharynx markers. (A) Probable location of the PAS136 pharynx phenotype allele is between 6 cM and 8 cM on LG.I relative to the genetic center of the chromosome (green circle) derived by mapping with DraI or EcoRI specific SNPs corresponding to DNA clones D1007, K02B12, B0205, and F58D5 (orange lines) and between 4.64 cM and 9.2 cM (red circle) using complementation with the deficiency strains MT2179, DC1079, KR2838 and SL536 with overlapping chromosomal deletions (blue lines). (B) pha-4 RNAi used a positive control for pharynx phenotypes, arrow shows lack of myo-2::GFP in most of the head. (C) lam-3 (T22A3.8) RNAi showing a phenotype similar to PAS136 with non-adherent cells (arrow). (D) blmp-1 (F25D7.3) RNAi has a less severe PAS136 phenotype (arrow denotes cell disconnected from the pharynx). (E) hmr-1 (W02B9.1) RNAi results in a Pun phenotype with diminished anterior pharynx cells (arrow). (F) Wild-type MH27 AJM-1 adherens junction antibody staining showing pharynx (ph) and intestine (it) localization. (G) PAS136 embryo with weak and disconnect AJM-1 staining in the pharynx (ph) and more normal AJM-1 in the intestine (it). (H) Wild-type Intermediate Filaments showing three sets of marginal cells (arrows). (I) PAS136 embryo with three sets of marginal cells (arrows). Bar is ∼10 µM.

    Techniques Used: Derivative Assay, Clone Assay, Positive Control, Laser Capture Microdissection, Staining

    15) Product Images from "Molecular Analysis of Antibiotic Resistance Determinants and Plasmids in Malaysian Isolates of Multidrug Resistant Klebsiella pneumoniae"

    Article Title: Molecular Analysis of Antibiotic Resistance Determinants and Plasmids in Malaysian Isolates of Multidrug Resistant Klebsiella pneumoniae

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0133654

    Dendrogram of EcoRI-digested plasmids from 27 transconjugants. 15 restriction profiles were identified (P1-P15). The dashed line represents the 80% similarity level used in cluster designation. Transconjugant plasmid ID, replicon and restriction profiles are shown.
    Figure Legend Snippet: Dendrogram of EcoRI-digested plasmids from 27 transconjugants. 15 restriction profiles were identified (P1-P15). The dashed line represents the 80% similarity level used in cluster designation. Transconjugant plasmid ID, replicon and restriction profiles are shown.

    Techniques Used: Plasmid Preparation

    16) Product Images from "Efficient Dual-Negative Selection for Bacterial Genome Editing"

    Article Title: Efficient Dual-Negative Selection for Bacterial Genome Editing

    Journal: bioRxiv

    doi: 10.1101/2020.03.03.974816

    An optimized method for genome editing in Salmonella enterica . a ) Map of suicide plasmid pFOK ( aphA , aminoglycoside phosphotransferase gene conferring resistance to kanamycin ; I-sceI gene encoding meganuclease; oriT , origin of conjugational transfer; P tetA , tetA promoter, R6K γ ori , pi-dependent origin of replication; sacB , levansucrase gene; tetR , tetracycline repressor gene; traJ , transcriptional activator for conjugational transfer genes; MCR, multi cloning region containing at least EcoRI, BamHI, SacI, XhoI and NotI). b-c ) Negative selection with SacB and I-SceI. b ) Mechanisms of negative selection for SacB and I-SceI, c ) Selection efficiency for various chromosomal loci ( foxA deletion, sitABCD deletion, ssrB point mutation and phoQ chimeric insertion [ 35 ]) using either SacB or I-SceI, or a combination of both. d-e ) Identification of recombination biases favoring one flanking region. d ) schematic representation of preferential recombination in the right flanking region. External primers (here primer 1 and 2) together with plasmid-specific primers (here primer oOPC-614 and oOPC-615) can be used to screen co-integrant clones to reveal such bias. e ) Recombination bias for foxA gene manipulation. PCR results of ex-conjugant screening using the primer pair 1 and oOPC-614 (left panel here oOPC-396/614) and primer 2 and oOPC-615 (right panel here oOPC-397/615). Rare ex-conjugants (here clone 5 and 10) with recombination in the non-preferred flanking region are used for subsequent counter-selection. f ) Timeframe with brief summary of daily steps.
    Figure Legend Snippet: An optimized method for genome editing in Salmonella enterica . a ) Map of suicide plasmid pFOK ( aphA , aminoglycoside phosphotransferase gene conferring resistance to kanamycin ; I-sceI gene encoding meganuclease; oriT , origin of conjugational transfer; P tetA , tetA promoter, R6K γ ori , pi-dependent origin of replication; sacB , levansucrase gene; tetR , tetracycline repressor gene; traJ , transcriptional activator for conjugational transfer genes; MCR, multi cloning region containing at least EcoRI, BamHI, SacI, XhoI and NotI). b-c ) Negative selection with SacB and I-SceI. b ) Mechanisms of negative selection for SacB and I-SceI, c ) Selection efficiency for various chromosomal loci ( foxA deletion, sitABCD deletion, ssrB point mutation and phoQ chimeric insertion [ 35 ]) using either SacB or I-SceI, or a combination of both. d-e ) Identification of recombination biases favoring one flanking region. d ) schematic representation of preferential recombination in the right flanking region. External primers (here primer 1 and 2) together with plasmid-specific primers (here primer oOPC-614 and oOPC-615) can be used to screen co-integrant clones to reveal such bias. e ) Recombination bias for foxA gene manipulation. PCR results of ex-conjugant screening using the primer pair 1 and oOPC-614 (left panel here oOPC-396/614) and primer 2 and oOPC-615 (right panel here oOPC-397/615). Rare ex-conjugants (here clone 5 and 10) with recombination in the non-preferred flanking region are used for subsequent counter-selection. f ) Timeframe with brief summary of daily steps.

    Techniques Used: Plasmid Preparation, Clone Assay, Selection, Mutagenesis, Polymerase Chain Reaction

    17) Product Images from "Methylase-assisted subcloning for high throughput BioBrick assembly"

    Article Title: Methylase-assisted subcloning for high throughput BioBrick assembly

    Journal: PeerJ

    doi: 10.7717/peerj.9841

    Model plasmids used in this study. The lacI-Ptac-lacO insert (A) includes a promoter that is somewhat leaky at high copy number. The IMBB2.4-pUC57-mini backbone (A–B), hereafter abbreviated pUC, is BioBrick-compatible and also includes an NsiI site downstream of PstI ( Matsumura, 2017 ). The tagRFP reporter (B) protein can cause colonies to turn visibly pink, but only when the gene encoding it is subcloned downstream of a leaky or constitutive promoter. RP4 oriT-pUC-cat (C) is a BioBrick compatible plasmid that confers resistance to chloramphenicol instead of ampicillin. RP4 oriT serves as a small stuffer in these experiments. In this study this latter plasmid is used only as a recipient plasmid (destination vector) for 3A assembly. Five expression vectors for production of recombinant DNA methyltransferases were constructed for this study. The version that expresses M.Ocy1ORF8430P, a putative ortholog of M.SpeI, is shown (D). The others are similar in design but express M.XbaI, M.EcoRI, M.PstI or M.AvaIII instead. Each plasmid utilizes the low copy number p15A origin (pACYC) and confers resistance to spectinomycin and is thus compatible with pUC plasmids that impart resistance to ampicillin, chloramphenicol or kanamycin. The DNA methyltransferase expression vectors do not contain any of the restriction sites employed in BioBrick assembly protocols (EcoRI, XbaI, SpeI or PstI), so they will not produce restriction fragments that ligate to those that are desired.
    Figure Legend Snippet: Model plasmids used in this study. The lacI-Ptac-lacO insert (A) includes a promoter that is somewhat leaky at high copy number. The IMBB2.4-pUC57-mini backbone (A–B), hereafter abbreviated pUC, is BioBrick-compatible and also includes an NsiI site downstream of PstI ( Matsumura, 2017 ). The tagRFP reporter (B) protein can cause colonies to turn visibly pink, but only when the gene encoding it is subcloned downstream of a leaky or constitutive promoter. RP4 oriT-pUC-cat (C) is a BioBrick compatible plasmid that confers resistance to chloramphenicol instead of ampicillin. RP4 oriT serves as a small stuffer in these experiments. In this study this latter plasmid is used only as a recipient plasmid (destination vector) for 3A assembly. Five expression vectors for production of recombinant DNA methyltransferases were constructed for this study. The version that expresses M.Ocy1ORF8430P, a putative ortholog of M.SpeI, is shown (D). The others are similar in design but express M.XbaI, M.EcoRI, M.PstI or M.AvaIII instead. Each plasmid utilizes the low copy number p15A origin (pACYC) and confers resistance to spectinomycin and is thus compatible with pUC plasmids that impart resistance to ampicillin, chloramphenicol or kanamycin. The DNA methyltransferase expression vectors do not contain any of the restriction sites employed in BioBrick assembly protocols (EcoRI, XbaI, SpeI or PstI), so they will not produce restriction fragments that ligate to those that are desired.

    Techniques Used: Plasmid Preparation, Expressing, Recombinant, Construct, Low Copy Number

    Conventional subcloning of BioBrick-compatible parts. Recipient (1) and donor (2) plasmids both contain inserts bound by the same restriction sites (E = EcoRI, X = XbaI, S = SpeI, P = PstI). The recipient plasmid (1) is cut with SpeI and PstI, releasing a short stuffer fragment (or “snippet,” 3); the donor (2) is separately cut with XbaI and PstI, so that insert (5) is released from plasmid fragment (6). The fragments from both digests (3-6) are separated by agarose gel electrophoresis. The desired recipient fragment (4) and insert (5) are excised from the gel and subsequently purified; the unwanted stuffer (3) and donor plasmid fragment (6) are left in the gels, which are thrown away. The recipient fragment (4) and insert (5) are ligated together forming three products: the recipient plasmid homodimer (7), the insert homodimer (8) and desired insert-recipient plasmid heterodimer (9). Large inverted repeats (7-8) cannot replicate stably in vivo so the desired construct (9) is the only product capable of conferring antibiotic selection if the digests and ligations were efficient.
    Figure Legend Snippet: Conventional subcloning of BioBrick-compatible parts. Recipient (1) and donor (2) plasmids both contain inserts bound by the same restriction sites (E = EcoRI, X = XbaI, S = SpeI, P = PstI). The recipient plasmid (1) is cut with SpeI and PstI, releasing a short stuffer fragment (or “snippet,” 3); the donor (2) is separately cut with XbaI and PstI, so that insert (5) is released from plasmid fragment (6). The fragments from both digests (3-6) are separated by agarose gel electrophoresis. The desired recipient fragment (4) and insert (5) are excised from the gel and subsequently purified; the unwanted stuffer (3) and donor plasmid fragment (6) are left in the gels, which are thrown away. The recipient fragment (4) and insert (5) are ligated together forming three products: the recipient plasmid homodimer (7), the insert homodimer (8) and desired insert-recipient plasmid heterodimer (9). Large inverted repeats (7-8) cannot replicate stably in vivo so the desired construct (9) is the only product capable of conferring antibiotic selection if the digests and ligations were efficient.

    Techniques Used: Subcloning, Plasmid Preparation, Agarose Gel Electrophoresis, Purification, Stable Transfection, In Vivo, Construct, Selection

    4R/2M (PstI) BioBrick assembly. The EcoRI site of the recipient plasmid (1) and SpeI site of the insert (2) are methylated in vivo. The recipient plasmid (1) is digested with SpeI and PstI, so that it releases a short 18 bp stuffer (or “snippet”, 4). The donor plasmid (2) is separately digested with XbaI and PstI, producing the desired insert (5) and the undesired donor plasmid fragment (6). The restriction enzymes are heat-killed, the digestion products are mixed and reacted with T4 DNA ligase, forming three sets of ligation products: parental-plasmids (1-2), homodimers (7-9) and heterodimers (10-13). The 36 bp snippet homodimer is not shown, nor are trimer, tetramer and other higher order products. The homodimer products (7-9) are large perfect inverted repeats, which are not expected to replicate efficiently in vivo. Moreover, none of the undesired parental (1-2), homodimer (7-9) or heterodimers (10-11) are resistant to subsequent digestion with EcoRI and SpeI. Only the desired insert/recipient recombinant plasmid (13) retains its ability to transform E. coli .
    Figure Legend Snippet: 4R/2M (PstI) BioBrick assembly. The EcoRI site of the recipient plasmid (1) and SpeI site of the insert (2) are methylated in vivo. The recipient plasmid (1) is digested with SpeI and PstI, so that it releases a short 18 bp stuffer (or “snippet”, 4). The donor plasmid (2) is separately digested with XbaI and PstI, producing the desired insert (5) and the undesired donor plasmid fragment (6). The restriction enzymes are heat-killed, the digestion products are mixed and reacted with T4 DNA ligase, forming three sets of ligation products: parental-plasmids (1-2), homodimers (7-9) and heterodimers (10-13). The 36 bp snippet homodimer is not shown, nor are trimer, tetramer and other higher order products. The homodimer products (7-9) are large perfect inverted repeats, which are not expected to replicate efficiently in vivo. Moreover, none of the undesired parental (1-2), homodimer (7-9) or heterodimers (10-11) are resistant to subsequent digestion with EcoRI and SpeI. Only the desired insert/recipient recombinant plasmid (13) retains its ability to transform E. coli .

    Techniques Used: Plasmid Preparation, Methylation, In Vivo, Ligation, Recombinant

    18) Product Images from "Methylase-assisted subcloning for high throughput BioBrick assembly"

    Article Title: Methylase-assisted subcloning for high throughput BioBrick assembly

    Journal: PeerJ

    doi: 10.7717/peerj.9841

    Model plasmids used in this study. The lacI-Ptac-lacO insert (A) includes a promoter that is somewhat leaky at high copy number. The IMBB2.4-pUC57-mini backbone (A–B), hereafter abbreviated pUC, is BioBrick-compatible and also includes an NsiI site downstream of PstI ( Matsumura, 2017 ). The tagRFP reporter (B) protein can cause colonies to turn visibly pink, but only when the gene encoding it is subcloned downstream of a leaky or constitutive promoter. RP4 oriT-pUC-cat (C) is a BioBrick compatible plasmid that confers resistance to chloramphenicol instead of ampicillin. RP4 oriT serves as a small stuffer in these experiments. In this study this latter plasmid is used only as a recipient plasmid (destination vector) for 3A assembly. Five expression vectors for production of recombinant DNA methyltransferases were constructed for this study. The version that expresses M.Ocy1ORF8430P, a putative ortholog of M.SpeI, is shown (D). The others are similar in design but express M.XbaI, M.EcoRI, M.PstI or M.AvaIII instead. Each plasmid utilizes the low copy number p15A origin (pACYC) and confers resistance to spectinomycin and is thus compatible with pUC plasmids that impart resistance to ampicillin, chloramphenicol or kanamycin. The DNA methyltransferase expression vectors do not contain any of the restriction sites employed in BioBrick assembly protocols (EcoRI, XbaI, SpeI or PstI), so they will not produce restriction fragments that ligate to those that are desired.
    Figure Legend Snippet: Model plasmids used in this study. The lacI-Ptac-lacO insert (A) includes a promoter that is somewhat leaky at high copy number. The IMBB2.4-pUC57-mini backbone (A–B), hereafter abbreviated pUC, is BioBrick-compatible and also includes an NsiI site downstream of PstI ( Matsumura, 2017 ). The tagRFP reporter (B) protein can cause colonies to turn visibly pink, but only when the gene encoding it is subcloned downstream of a leaky or constitutive promoter. RP4 oriT-pUC-cat (C) is a BioBrick compatible plasmid that confers resistance to chloramphenicol instead of ampicillin. RP4 oriT serves as a small stuffer in these experiments. In this study this latter plasmid is used only as a recipient plasmid (destination vector) for 3A assembly. Five expression vectors for production of recombinant DNA methyltransferases were constructed for this study. The version that expresses M.Ocy1ORF8430P, a putative ortholog of M.SpeI, is shown (D). The others are similar in design but express M.XbaI, M.EcoRI, M.PstI or M.AvaIII instead. Each plasmid utilizes the low copy number p15A origin (pACYC) and confers resistance to spectinomycin and is thus compatible with pUC plasmids that impart resistance to ampicillin, chloramphenicol or kanamycin. The DNA methyltransferase expression vectors do not contain any of the restriction sites employed in BioBrick assembly protocols (EcoRI, XbaI, SpeI or PstI), so they will not produce restriction fragments that ligate to those that are desired.

    Techniques Used: Plasmid Preparation, Expressing, Recombinant, Construct, Low Copy Number

    Conventional subcloning of BioBrick-compatible parts. Recipient (1) and donor (2) plasmids both contain inserts bound by the same restriction sites (E = EcoRI, X = XbaI, S = SpeI, P = PstI). The recipient plasmid (1) is cut with SpeI and PstI, releasing a short stuffer fragment (or “snippet,” 3); the donor (2) is separately cut with XbaI and PstI, so that insert (5) is released from plasmid fragment (6). The fragments from both digests (3-6) are separated by agarose gel electrophoresis. The desired recipient fragment (4) and insert (5) are excised from the gel and subsequently purified; the unwanted stuffer (3) and donor plasmid fragment (6) are left in the gels, which are thrown away. The recipient fragment (4) and insert (5) are ligated together forming three products: the recipient plasmid homodimer (7), the insert homodimer (8) and desired insert-recipient plasmid heterodimer (9). Large inverted repeats (7-8) cannot replicate stably in vivo so the desired construct (9) is the only product capable of conferring antibiotic selection if the digests and ligations were efficient.
    Figure Legend Snippet: Conventional subcloning of BioBrick-compatible parts. Recipient (1) and donor (2) plasmids both contain inserts bound by the same restriction sites (E = EcoRI, X = XbaI, S = SpeI, P = PstI). The recipient plasmid (1) is cut with SpeI and PstI, releasing a short stuffer fragment (or “snippet,” 3); the donor (2) is separately cut with XbaI and PstI, so that insert (5) is released from plasmid fragment (6). The fragments from both digests (3-6) are separated by agarose gel electrophoresis. The desired recipient fragment (4) and insert (5) are excised from the gel and subsequently purified; the unwanted stuffer (3) and donor plasmid fragment (6) are left in the gels, which are thrown away. The recipient fragment (4) and insert (5) are ligated together forming three products: the recipient plasmid homodimer (7), the insert homodimer (8) and desired insert-recipient plasmid heterodimer (9). Large inverted repeats (7-8) cannot replicate stably in vivo so the desired construct (9) is the only product capable of conferring antibiotic selection if the digests and ligations were efficient.

    Techniques Used: Subcloning, Plasmid Preparation, Agarose Gel Electrophoresis, Purification, Stable Transfection, In Vivo, Construct, Selection

    4R/2M (PstI) BioBrick assembly. The EcoRI site of the recipient plasmid (1) and SpeI site of the insert (2) are methylated in vivo. The recipient plasmid (1) is digested with SpeI and PstI, so that it releases a short 18 bp stuffer (or “snippet”, 4). The donor plasmid (2) is separately digested with XbaI and PstI, producing the desired insert (5) and the undesired donor plasmid fragment (6). The restriction enzymes are heat-killed, the digestion products are mixed and reacted with T4 DNA ligase, forming three sets of ligation products: parental-plasmids (1-2), homodimers (7-9) and heterodimers (10-13). The 36 bp snippet homodimer is not shown, nor are trimer, tetramer and other higher order products. The homodimer products (7-9) are large perfect inverted repeats, which are not expected to replicate efficiently in vivo. Moreover, none of the undesired parental (1-2), homodimer (7-9) or heterodimers (10-11) are resistant to subsequent digestion with EcoRI and SpeI. Only the desired insert/recipient recombinant plasmid (13) retains its ability to transform E. coli .
    Figure Legend Snippet: 4R/2M (PstI) BioBrick assembly. The EcoRI site of the recipient plasmid (1) and SpeI site of the insert (2) are methylated in vivo. The recipient plasmid (1) is digested with SpeI and PstI, so that it releases a short 18 bp stuffer (or “snippet”, 4). The donor plasmid (2) is separately digested with XbaI and PstI, producing the desired insert (5) and the undesired donor plasmid fragment (6). The restriction enzymes are heat-killed, the digestion products are mixed and reacted with T4 DNA ligase, forming three sets of ligation products: parental-plasmids (1-2), homodimers (7-9) and heterodimers (10-13). The 36 bp snippet homodimer is not shown, nor are trimer, tetramer and other higher order products. The homodimer products (7-9) are large perfect inverted repeats, which are not expected to replicate efficiently in vivo. Moreover, none of the undesired parental (1-2), homodimer (7-9) or heterodimers (10-11) are resistant to subsequent digestion with EcoRI and SpeI. Only the desired insert/recipient recombinant plasmid (13) retains its ability to transform E. coli .

    Techniques Used: Plasmid Preparation, Methylation, In Vivo, Ligation, Recombinant

    Related Articles

    Labeling:

    Article Title: The Chd1 chromatin remodeler can sense both entry and exit sides of the nucleosome
    Article Snippet: Restriction digestion An EcoRI restriction cut site was introduced within the 601 positioning sequence by PCR amplification, 10–15 bp from the left edge of the 601 sequence. .. Digestions of 150 nM fluorescently labeled [FAM]0N70[LacO-11R,Cy5] nucleosomes were carried out using 2 U/μl of EcoRI-HF (NEB) in 1×CutSmart buffer at 30°C. .. After pre-incubation of Chd1 (100 nM), nucleosomes (150 nM) and ±LacI (1.2 μM) for 5 min, EcoR1-HF was added for 2 min to digest free DNA, followed by ATP addition to a final concentration of 2.5 mM.

    Incubation:

    Article Title: DNA translocase repositions a nucleosome by the lane-switch mechanism
    Article Snippet: Next, we added 1 μL of RNase I (M0243, New England BioLabs) to reactions in 1 x NEBuffer 3.1 (B7003, New England BioLabs) and incubated for 30 minutes at 37°C to remove the transcripts. .. Then, we exchanged the buffer for the CutSmart buffer (B7204 New England BioLabs) using a 10K MWCO Amicon® Ultra-0.5 Centrifugal Filter (UFC510024, Merck), added BssSI-v2 (R0680, New England BioLabs) or EcoRI-HF (R3101, New England BioLabs), and incubated for 15 minutes at 37 °C. .. The digested products were extracted using a phenol:chloroform:isoamyl (25:24:1) solution (Nakalai Tesque) and run on 1% agarose gel.

    Article Title: An exogenous chloroplast genome for complex sequence manipulation in algae
    Article Snippet: Approximately 107 cells were collected, suspended in lysis buffer (150 mM Tris–HCl, pH = 7.5, 200 mM NaCl, 20 mM EDTA and 1% SDS), incubated for 1 h at 37°C, extracted once with phenol/chloroform (1:1) and twice with chloroform, ethanol precipitated and resuspended in TE buffer (10 mM Tris–HCl, pH = 7.4, 1 mM EDTA, 50 µg/ml RNase). .. For digestions, 10 µg of total algae genomic DNA or 250 ng of the exogenous chloroplast genome DNA isolated from bacteria were incubated with EcoRI-HF or NdeI (New England Biolabs; Ipswich, MA, USA) for 3 h at 37°C in a total volume of 50 µl. .. Digestion products were separated on a 0.7% agarose gel run in TAE.

    Transformation Assay:

    Article Title: Genetic transformation of Spizellomyces punctatus, a resource for studying chytrid biology and evolutionary cell biology
    Article Snippet: Identification of T-DNA insertion sites by inverse PCR. .. 2.5 µg of genomic DNA of wild type (WT) and the four transformed strains of Spizellomyces (EM20C-1,2,3,4) was digested in a final volume of 50μL with 100U of EcoRI-HF (NEB R3101S) or HindIII-HF (NEB R3104S) for 24 hr at 37°C. .. After assessing the quality of the digestion by gel electrophoresis, the reaction was heat inactivated and 48µL of the digested DNA was incubated with 1µL of T4 ligase (400u/µL) for 48 hr at 4°C.

    Amplification:

    Article Title: Engineered viral DNA polymerase with enhanced DNA amplification capacity: a proof-of-concept of isothermal amplification of damaged DNA
    Article Snippet: The difference between B35DNAP and B35-HhH amplification performance was tested by a paired t-test (Prism GraphPad Software). .. To analyse the fidelity of B35DNAP, we treated a fraction of the amplified DNA product with EcoRI-HF and DpnI (New England Biolabs) for 2 h at 37 °C. .. The digested products were purified using the QIAquick PCR Purification Kit (Qiagen N.V.), and 170 ng of pUC19 linear plasmid was circularised with T4 DNA ligase (New England Biolabs) in a final volume of 100 μL.

    Plasmid Preparation:

    Article Title: Depurination of colibactin-derived interstrand cross-links
    Article Snippet: DNA Cross-linking Assays Linearized pUC19 DNA was used for all DNA cross-linking assays. .. To prepare the linearized DNA, the 2686 bp pUC19 vector (New England Biolabs®) was linearized with 20 units/µg EcoRI-HF® (New England Biolabs®) and the linearized DNA was purified using a PCR clean-up kit (New England Biolabs®), eluted into 10 mM Tris (pH 8.0), and quantified using a nanodrop. .. For each reaction with E.coli , 800 ng of linearized pUC19 DNA was added to 200 µL (6.2 µM base pairs) of M9-CA medium inoculated with 1.2 × 107 bacteria pre-grown to exponential phase in the M9-CA medium.

    Purification:

    Article Title: Depurination of colibactin-derived interstrand cross-links
    Article Snippet: DNA Cross-linking Assays Linearized pUC19 DNA was used for all DNA cross-linking assays. .. To prepare the linearized DNA, the 2686 bp pUC19 vector (New England Biolabs®) was linearized with 20 units/µg EcoRI-HF® (New England Biolabs®) and the linearized DNA was purified using a PCR clean-up kit (New England Biolabs®), eluted into 10 mM Tris (pH 8.0), and quantified using a nanodrop. .. For each reaction with E.coli , 800 ng of linearized pUC19 DNA was added to 200 µL (6.2 µM base pairs) of M9-CA medium inoculated with 1.2 × 107 bacteria pre-grown to exponential phase in the M9-CA medium.

    Polymerase Chain Reaction:

    Article Title: Depurination of colibactin-derived interstrand cross-links
    Article Snippet: DNA Cross-linking Assays Linearized pUC19 DNA was used for all DNA cross-linking assays. .. To prepare the linearized DNA, the 2686 bp pUC19 vector (New England Biolabs®) was linearized with 20 units/µg EcoRI-HF® (New England Biolabs®) and the linearized DNA was purified using a PCR clean-up kit (New England Biolabs®), eluted into 10 mM Tris (pH 8.0), and quantified using a nanodrop. .. For each reaction with E.coli , 800 ng of linearized pUC19 DNA was added to 200 µL (6.2 µM base pairs) of M9-CA medium inoculated with 1.2 × 107 bacteria pre-grown to exponential phase in the M9-CA medium.

    Isolation:

    Article Title: An exogenous chloroplast genome for complex sequence manipulation in algae
    Article Snippet: Approximately 107 cells were collected, suspended in lysis buffer (150 mM Tris–HCl, pH = 7.5, 200 mM NaCl, 20 mM EDTA and 1% SDS), incubated for 1 h at 37°C, extracted once with phenol/chloroform (1:1) and twice with chloroform, ethanol precipitated and resuspended in TE buffer (10 mM Tris–HCl, pH = 7.4, 1 mM EDTA, 50 µg/ml RNase). .. For digestions, 10 µg of total algae genomic DNA or 250 ng of the exogenous chloroplast genome DNA isolated from bacteria were incubated with EcoRI-HF or NdeI (New England Biolabs; Ipswich, MA, USA) for 3 h at 37°C in a total volume of 50 µl. .. Digestion products were separated on a 0.7% agarose gel run in TAE.

    Generated:

    Article Title: Multiple Pairwise Analysis of Non-homologous Centromere Coupling Reveals Preferential Chromosome Size-Dependent Interactions and a Role for Bouquet Formation in Establishing the Interaction Pattern
    Article Snippet: .. For the double digestion, EcoRI-HF and MfeI-HF (NEB) generated cohesive ends that were compatible, yet recognizing slightly different sequences (GAATTC and CAATTG respectively). .. They were selected due to their extremely low star activity, extended enzymatic half-life suitable for overnight digestion and stronger activity in samples of lower purity, making them appropriate for digestion of crosslinked chromatin.

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    New England Biolabs ecori hf
    Mapping T-DNA genomic insertion sites with inverse PCR. ( A ) Diagram of T-DNA integration and the location of PCR/sequencing primers and restriction sites used for inverse PCR (invPCR). We only show primers adjacent to the left border (LB) because they consistently amplified for all transformants, unlike the primers adjacent to the right border. ( B ) Example of amplification by invPCR of the LB-genome border after <t>EcoRI</t> genomic digestion and ligation for an untransformed strain (WT), four independent transformants and non-template control. ( C ) Amplification by invPCR of the LB-genome border after HindIII genomic digestion and ligation. T-DNA location for all transformants was confirmed by two independent biological replicates (i.e. independent genomic extractions, ligation and invPCR). ( D ) T-DNA insertion sites in four independent transformants of Spizellomyces . In strain <t>EM20C-3,</t> invPCR for EcoRI indicated LB is located toward SPPG_02523, while invPCR for HindIII shows same insertion site but with an inverted direction. The divergent invPCR results might represent an insertion of a tandem inverted T-DNA. ( E ) Three of the four strains (EM20C-2,3,4) have similar tdTomato fluorescence levels as determined by flow cytometry.
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    Mapping T-DNA genomic insertion sites with inverse PCR. ( A ) Diagram of T-DNA integration and the location of PCR/sequencing primers and restriction sites used for inverse PCR (invPCR). We only show primers adjacent to the left border (LB) because they consistently amplified for all transformants, unlike the primers adjacent to the right border. ( B ) Example of amplification by invPCR of the LB-genome border after EcoRI genomic digestion and ligation for an untransformed strain (WT), four independent transformants and non-template control. ( C ) Amplification by invPCR of the LB-genome border after HindIII genomic digestion and ligation. T-DNA location for all transformants was confirmed by two independent biological replicates (i.e. independent genomic extractions, ligation and invPCR). ( D ) T-DNA insertion sites in four independent transformants of Spizellomyces . In strain EM20C-3, invPCR for EcoRI indicated LB is located toward SPPG_02523, while invPCR for HindIII shows same insertion site but with an inverted direction. The divergent invPCR results might represent an insertion of a tandem inverted T-DNA. ( E ) Three of the four strains (EM20C-2,3,4) have similar tdTomato fluorescence levels as determined by flow cytometry.

    Journal: eLife

    Article Title: Genetic transformation of Spizellomyces punctatus, a resource for studying chytrid biology and evolutionary cell biology

    doi: 10.7554/eLife.52741

    Figure Lengend Snippet: Mapping T-DNA genomic insertion sites with inverse PCR. ( A ) Diagram of T-DNA integration and the location of PCR/sequencing primers and restriction sites used for inverse PCR (invPCR). We only show primers adjacent to the left border (LB) because they consistently amplified for all transformants, unlike the primers adjacent to the right border. ( B ) Example of amplification by invPCR of the LB-genome border after EcoRI genomic digestion and ligation for an untransformed strain (WT), four independent transformants and non-template control. ( C ) Amplification by invPCR of the LB-genome border after HindIII genomic digestion and ligation. T-DNA location for all transformants was confirmed by two independent biological replicates (i.e. independent genomic extractions, ligation and invPCR). ( D ) T-DNA insertion sites in four independent transformants of Spizellomyces . In strain EM20C-3, invPCR for EcoRI indicated LB is located toward SPPG_02523, while invPCR for HindIII shows same insertion site but with an inverted direction. The divergent invPCR results might represent an insertion of a tandem inverted T-DNA. ( E ) Three of the four strains (EM20C-2,3,4) have similar tdTomato fluorescence levels as determined by flow cytometry.

    Article Snippet: 2.5 µg of genomic DNA of wild type (WT) and the four transformed strains of Spizellomyces (EM20C-1,2,3,4) was digested in a final volume of 50μL with 100U of EcoRI-HF (NEB R3101S) or HindIII-HF (NEB R3104S) for 24 hr at 37°C.

    Techniques: Inverse PCR, Polymerase Chain Reaction, Sequencing, Amplification, Ligation, Fluorescence, Flow Cytometry

    Restriction enzyme digestion assay. (A) The DNA sequence map of substrates used in the assay. The T7 RNAP stall sites where the translocase first encounter adenine are marked by the black arrows. BssSI and EcoRI restriction sites are marked by the blue and red arrows, respectively. (B) Cartoons of the experimental setup. At the pre-transcription stage, the BssSI site, but not the EcoRI site, is occluded by the histone core complex. On the other hand, if the histone core complex repositions upon transcription according to the lane-switch mechanism, the EcoRI site, but not the BssSI site, is occluded by the histone core complex. (C) Images of 1% agarose gel on which the EcoRI-digested products run. The DNA substrates were digested at the pre- and post-transcription stages. (D) Plot showing the intensity of the undigested band at pre-transcription stage divided by that of the post-transcription stage (Δ occlusion ). The assay was repeated using naked DNA (Naked), nucleosome reconstituted DNA (Nucleosome), and the naked DNA with histone core complexes in solution (Octamer).

    Journal: bioRxiv

    Article Title: DNA translocase repositions a nucleosome by the lane-switch mechanism

    doi: 10.1101/2021.02.15.431322

    Figure Lengend Snippet: Restriction enzyme digestion assay. (A) The DNA sequence map of substrates used in the assay. The T7 RNAP stall sites where the translocase first encounter adenine are marked by the black arrows. BssSI and EcoRI restriction sites are marked by the blue and red arrows, respectively. (B) Cartoons of the experimental setup. At the pre-transcription stage, the BssSI site, but not the EcoRI site, is occluded by the histone core complex. On the other hand, if the histone core complex repositions upon transcription according to the lane-switch mechanism, the EcoRI site, but not the BssSI site, is occluded by the histone core complex. (C) Images of 1% agarose gel on which the EcoRI-digested products run. The DNA substrates were digested at the pre- and post-transcription stages. (D) Plot showing the intensity of the undigested band at pre-transcription stage divided by that of the post-transcription stage (Δ occlusion ). The assay was repeated using naked DNA (Naked), nucleosome reconstituted DNA (Nucleosome), and the naked DNA with histone core complexes in solution (Octamer).

    Article Snippet: Then, we exchanged the buffer for the CutSmart buffer (B7204 New England BioLabs) using a 10K MWCO Amicon® Ultra-0.5 Centrifugal Filter (UFC510024, Merck), added BssSI-v2 (R0680, New England BioLabs) or EcoRI-HF (R3101, New England BioLabs), and incubated for 15 minutes at 37 °C.

    Techniques: Sequencing, Agarose Gel Electrophoresis

    The images of 1% agarose gel on which the digested products run. The DNA substrates were digested by BssSI and EcoRI at the pre-and post-transcription stages. Depending on the substrates, the RNAP stalled at −54 bps (top), −29 bps (middle), and −14 bps (bottom), respectively. The assay was repeated using naked DNA (Naked), nucleosome reconstituted DNA (Nucleosome), and the naked DNA with histone core complexes in solution (Octamer). The marker is Quick-Load Purple 50 bp DNA Ladder (New England BioLabs; N0556S).

    Journal: bioRxiv

    Article Title: DNA translocase repositions a nucleosome by the lane-switch mechanism

    doi: 10.1101/2021.02.15.431322

    Figure Lengend Snippet: The images of 1% agarose gel on which the digested products run. The DNA substrates were digested by BssSI and EcoRI at the pre-and post-transcription stages. Depending on the substrates, the RNAP stalled at −54 bps (top), −29 bps (middle), and −14 bps (bottom), respectively. The assay was repeated using naked DNA (Naked), nucleosome reconstituted DNA (Nucleosome), and the naked DNA with histone core complexes in solution (Octamer). The marker is Quick-Load Purple 50 bp DNA Ladder (New England BioLabs; N0556S).

    Article Snippet: Then, we exchanged the buffer for the CutSmart buffer (B7204 New England BioLabs) using a 10K MWCO Amicon® Ultra-0.5 Centrifugal Filter (UFC510024, Merck), added BssSI-v2 (R0680, New England BioLabs) or EcoRI-HF (R3101, New England BioLabs), and incubated for 15 minutes at 37 °C.

    Techniques: Agarose Gel Electrophoresis, Marker

    Exit side DNA defines the extent that Chd1 slides nucleosomes in the presence of Lac repressor. ( A ) Nucleosomes are dynamically shifted back-and-forth by Chd1 in the presence of LacI. End-positioned 0N70 nucleosomes containing a LacO(−11) site on the 70 bp side and an EcoRI cut site just inside the 0 bp side were digested by EcoRI and monitored by SDS-PAGE. The slower rate of digestion in the absence of ATP (gray) demonstrates that the EcoRI site is initially buried. In the presence of Chd1 and ATP, the same fraction of nucleosomal DNA becomes cleaved in the presence (filled circle) or absence (open circle) of LacI, demonstrating dynamic repositioning by Chd1 in the presence of LacI. Based on single exponential fits to the data, the sliding rate in the presence of LacI was calculated to be 7-fold slower than in the absence of LacI. Error bars indicate the standard deviations from five or more independent experiments. ( B ) Comparison of nucleosome sliding reactions carried out in the absence and presence of LacI, using (−10)N80[LacO-11R] substrates. Time points for these experiments were 0, 1, 4, 16, 64 min. ( C ) Comparison of the preferred distributions of nucleosome positions for 0N80 and (−10)N80 nucleosomes when Chd1 sliding was carried out in the presence of LacI. White peaks show zero time points and gray peaks are the nucleosome positions at 64 min time points.

    Journal: Nucleic Acids Research

    Article Title: The Chd1 chromatin remodeler can sense both entry and exit sides of the nucleosome

    doi: 10.1093/nar/gkw406

    Figure Lengend Snippet: Exit side DNA defines the extent that Chd1 slides nucleosomes in the presence of Lac repressor. ( A ) Nucleosomes are dynamically shifted back-and-forth by Chd1 in the presence of LacI. End-positioned 0N70 nucleosomes containing a LacO(−11) site on the 70 bp side and an EcoRI cut site just inside the 0 bp side were digested by EcoRI and monitored by SDS-PAGE. The slower rate of digestion in the absence of ATP (gray) demonstrates that the EcoRI site is initially buried. In the presence of Chd1 and ATP, the same fraction of nucleosomal DNA becomes cleaved in the presence (filled circle) or absence (open circle) of LacI, demonstrating dynamic repositioning by Chd1 in the presence of LacI. Based on single exponential fits to the data, the sliding rate in the presence of LacI was calculated to be 7-fold slower than in the absence of LacI. Error bars indicate the standard deviations from five or more independent experiments. ( B ) Comparison of nucleosome sliding reactions carried out in the absence and presence of LacI, using (−10)N80[LacO-11R] substrates. Time points for these experiments were 0, 1, 4, 16, 64 min. ( C ) Comparison of the preferred distributions of nucleosome positions for 0N80 and (−10)N80 nucleosomes when Chd1 sliding was carried out in the presence of LacI. White peaks show zero time points and gray peaks are the nucleosome positions at 64 min time points.

    Article Snippet: Digestions of 150 nM fluorescently labeled [FAM]0N70[LacO-11R,Cy5] nucleosomes were carried out using 2 U/μl of EcoRI-HF (NEB) in 1×CutSmart buffer at 30°C.

    Techniques: SDS Page

    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: To prepare the linearized DNA, the 2686 bp pUC19 vector (New England Biolabs®) was linearized with 20 units/µg EcoRI-HF® (New England Biolabs®) and the linearized DNA was purified using a PCR clean-up kit (New England Biolabs®), eluted into 10 mM Tris (pH 8.0), and quantified using a nanodrop.

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