dra i  (New England Biolabs)


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    Name:
    DraI
    Description:
    DraI 10 000 units
    Catalog Number:
    R0129L
    Price:
    269
    Category:
    Restriction Enzymes
    Size:
    10 000 units
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    Structured Review

    New England Biolabs dra i
    DraI
    DraI 10 000 units
    https://www.bioz.com/result/dra i/product/New England Biolabs
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dra i - by Bioz Stars, 2021-06
    96/100 stars

    Images

    1) Product Images from "Promoterless Transposon Mutagenesis Drives Solid Cancers via Tumor Suppressor Inactivation"

    Article Title: Promoterless Transposon Mutagenesis Drives Solid Cancers via Tumor Suppressor Inactivation

    Journal: bioRxiv

    doi: 10.1101/2020.08.17.254565

    Transgenic SB-T2/Onc2.3 founder mice. ( a ) Plasmid map of pT2/Onc2.3-MBM102, denoting essential features and base pair position in parenthesis, used to create the T2/Onc2.3 high-copy transgenic lines. ( b ) Southern blot of T2/Onc2.3 founder mice using a probe corresponding to the En2 -SA ( Engrailed 2 splice acceptor) element within the SB-T2/SB-GT allele construct that also cross hybridizes to the endogenous En2 mouse gene on chromosome 5. Genomic DNA digested to completion with Dra I restriction enzyme and detected with a radioisotope- 32 P labeled En2 -SA probe identifies the diploid endogenous En2 locus as a discrete 2.1 kb band and any all copies of the T2/SB-GT monomer that have been liberated from the their randomly inserted transgenic loci as a discrete 1.0 kb band. Mice with multi-copy concatemer alleles are identified as containing darker staining bands relative to the two copies of the En2 endogenous locus band. Blue arrows and dotted boxes denote animals selected for germ line breeding — five separate transgenic founders, three females (TG.14885, TG.14922, and TG.14942) and two males (TG.14913 and TG.14927), were selected for mating with wildtype C57BL/6J mice to confirm the germ line transmission of the SB-T/2SB-GT concatemer alleles to progeny. Mice denoted with blue dotted boxes produced transgenic carrier pups with a transgene copy numbers that differed between individuals, suggesting the presence of two or more independent transgenic concatemer integration sites. Mice denoted with red dotted boxes produced transgenic carrier pups with a transgene copy number that did not differ between individuals, suggesting that they resulted from a single transgenic concatemer integration site. Lines established from founders TG.14913, TG.14922, and TG.14942 were expanded and bred for further experiments. Bam HI restriction digested lambda (ƛ)-phage DNA is provided as a reference, a small amount was added to the radioisotope- 32 P labeling reaction with the En2 -SA probe to show detection on the developed Southern blot.
    Figure Legend Snippet: Transgenic SB-T2/Onc2.3 founder mice. ( a ) Plasmid map of pT2/Onc2.3-MBM102, denoting essential features and base pair position in parenthesis, used to create the T2/Onc2.3 high-copy transgenic lines. ( b ) Southern blot of T2/Onc2.3 founder mice using a probe corresponding to the En2 -SA ( Engrailed 2 splice acceptor) element within the SB-T2/SB-GT allele construct that also cross hybridizes to the endogenous En2 mouse gene on chromosome 5. Genomic DNA digested to completion with Dra I restriction enzyme and detected with a radioisotope- 32 P labeled En2 -SA probe identifies the diploid endogenous En2 locus as a discrete 2.1 kb band and any all copies of the T2/SB-GT monomer that have been liberated from the their randomly inserted transgenic loci as a discrete 1.0 kb band. Mice with multi-copy concatemer alleles are identified as containing darker staining bands relative to the two copies of the En2 endogenous locus band. Blue arrows and dotted boxes denote animals selected for germ line breeding — five separate transgenic founders, three females (TG.14885, TG.14922, and TG.14942) and two males (TG.14913 and TG.14927), were selected for mating with wildtype C57BL/6J mice to confirm the germ line transmission of the SB-T/2SB-GT concatemer alleles to progeny. Mice denoted with blue dotted boxes produced transgenic carrier pups with a transgene copy numbers that differed between individuals, suggesting the presence of two or more independent transgenic concatemer integration sites. Mice denoted with red dotted boxes produced transgenic carrier pups with a transgene copy number that did not differ between individuals, suggesting that they resulted from a single transgenic concatemer integration site. Lines established from founders TG.14913, TG.14922, and TG.14942 were expanded and bred for further experiments. Bam HI restriction digested lambda (ƛ)-phage DNA is provided as a reference, a small amount was added to the radioisotope- 32 P labeling reaction with the En2 -SA probe to show detection on the developed Southern blot.

    Techniques Used: Transgenic Assay, Mouse Assay, Plasmid Preparation, Southern Blot, Construct, Labeling, Staining, Transmission Assay, Produced

    Estimating SB transposon concatemer copy number from different SB strains. ( a ) Inverse image of EtBr stained agarose gel (1.2% TBE, run at 30 volts for 30 hours) containing genomic DNA digested with Dra I for 16 hours at 37 °C for Southern blot analysis. Molecular weight markers: left, lambda cut with Hin DIII; right, 100 bp ladder. ( b ) Southern blot demonstrating relative concatemer copy number differences between high-copy and low-copy SB transposon alleles in homozygous mice with unmobilized transposons. The En2 site provides a reference for a single copy, diploid gene.
    Figure Legend Snippet: Estimating SB transposon concatemer copy number from different SB strains. ( a ) Inverse image of EtBr stained agarose gel (1.2% TBE, run at 30 volts for 30 hours) containing genomic DNA digested with Dra I for 16 hours at 37 °C for Southern blot analysis. Molecular weight markers: left, lambda cut with Hin DIII; right, 100 bp ladder. ( b ) Southern blot demonstrating relative concatemer copy number differences between high-copy and low-copy SB transposon alleles in homozygous mice with unmobilized transposons. The En2 site provides a reference for a single copy, diploid gene.

    Techniques Used: Staining, Agarose Gel Electrophoresis, Southern Blot, Molecular Weight, Mouse Assay

    2) Product Images from "Analysis of Distant Communication on Defined Chromatin Templates In Vitro"

    Article Title: Analysis of Distant Communication on Defined Chromatin Templates In Vitro

    Journal: Methods in molecular biology (Clifton, N.J.)

    doi: 10.1007/978-1-60327-015-1_33

    Characterization of chromatin templates using restriction enzyme sensitivity assay. Chromatin was assembled on supercoiled pYP05 plasmid and digested with an excess of restriction enzymes Dra I and Bgl II. Then DNA was purified and analyzed in 1% agarose
    Figure Legend Snippet: Characterization of chromatin templates using restriction enzyme sensitivity assay. Chromatin was assembled on supercoiled pYP05 plasmid and digested with an excess of restriction enzymes Dra I and Bgl II. Then DNA was purified and analyzed in 1% agarose

    Techniques Used: Sensitive Assay, Plasmid Preparation, Purification

    3) Product Images from "Circular RNA biogenesis can proceed through an exon-containing lariat precursor"

    Article Title: Circular RNA biogenesis can proceed through an exon-containing lariat precursor

    Journal: eLife

    doi: 10.7554/eLife.07540

    Additional Dra I restriction digest PCR for Y-shaped intermediate product. Panel 2D with additional lanes indicating attempted amplification of the Y-shaped intermediate. As in 2D, primers used are indicated above the gel and observed products are illustrated below. The off-target product is the higher molecular weight species (lane 13) and represents mispriming of the reverse primer to a downstream site in exon 3. DOI: http://dx.doi.org/10.7554/eLife.07540.010
    Figure Legend Snippet: Additional Dra I restriction digest PCR for Y-shaped intermediate product. Panel 2D with additional lanes indicating attempted amplification of the Y-shaped intermediate. As in 2D, primers used are indicated above the gel and observed products are illustrated below. The off-target product is the higher molecular weight species (lane 13) and represents mispriming of the reverse primer to a downstream site in exon 3. DOI: http://dx.doi.org/10.7554/eLife.07540.010

    Techniques Used: Polymerase Chain Reaction, Amplification, Molecular Weight

    4) Product Images from "Characterization of DNA synthesis catalyzed by bacteriophage T4 replication complexes reconstituted on synthetic circular substrates"

    Article Title: Characterization of DNA synthesis catalyzed by bacteriophage T4 replication complexes reconstituted on synthetic circular substrates

    Journal: Nucleic Acids Research

    doi:

    Effect of an abasic site on DNA synthesis by reconstituted phage T4 replication complexes. ( A ) An abasic site blocks DNA synthesis by T4 DNA polymerase holoenzyme. 32 P-end-labeled 21mer #7 annealed with 120mer #3 (lanes 1, 3, 5, 7, 9, 11, 13 and 15) or with the same oligomer containing an abasic site at position 99 (120mer #4) (lanes 2, 4, 6, 8, 10, 12, 14 and 16) was incubated with either T4 gp43 (lanes 5, 6, 13 and 14) or the exonuclease-deficient gp43D219A (lanes 7, 8, 15 and 16) in the presence of gp44/62, gp45 and gp32 for the indicated times, and the products were separated in a 10% polyacrylamide gel + 6 M urea. Note that in the substrate with an abasic site, the first nucleotide to be incorporated into the 32 P-end-labeled 21mer is opposite the abasic site. ( B ) Block to lagging-strand synthesis imposed by the abasic site. Products of DNA synthesized by reconstituted T4 replication complexes in the presence of [α- 32 P]dCTP and 240mer circular ssDNA annealed with the 120mer ssDNA without (lanes 1 and 2) or with (lanes 3 and 4) abasic site were digested (lanes 2 and 4) or not (lanes 1 and 3) by Dra I and separated in a 6% polyacrylamide gel + 6 M urea along with marker DNA. Note that the 240mer circular ssDNA has a unique Dra I site. Upon Dra I cleavage of the replication products, the major 240mer fragment is formed along with other fragments that are located between the Dra I site and either the 3′ or 5′ ends of Okazaki fragments. Arrows show positions of the major 240-nt Dra I fragment and the 126-nt fragment located between the Dra I site and the 3′ end of the nascent fragment terminated by the abasic site. ( C ) DNA products were synthesized by reconstituted T4 replication complexes in the presence of [α- 32 P]dTTP and of 240mer circular ssDNA annealed with 120mer ssDNA without (lanes 1–3) or with (lanes 4–6) an abasic site, and separated in a 0.6% alkali gel.
    Figure Legend Snippet: Effect of an abasic site on DNA synthesis by reconstituted phage T4 replication complexes. ( A ) An abasic site blocks DNA synthesis by T4 DNA polymerase holoenzyme. 32 P-end-labeled 21mer #7 annealed with 120mer #3 (lanes 1, 3, 5, 7, 9, 11, 13 and 15) or with the same oligomer containing an abasic site at position 99 (120mer #4) (lanes 2, 4, 6, 8, 10, 12, 14 and 16) was incubated with either T4 gp43 (lanes 5, 6, 13 and 14) or the exonuclease-deficient gp43D219A (lanes 7, 8, 15 and 16) in the presence of gp44/62, gp45 and gp32 for the indicated times, and the products were separated in a 10% polyacrylamide gel + 6 M urea. Note that in the substrate with an abasic site, the first nucleotide to be incorporated into the 32 P-end-labeled 21mer is opposite the abasic site. ( B ) Block to lagging-strand synthesis imposed by the abasic site. Products of DNA synthesized by reconstituted T4 replication complexes in the presence of [α- 32 P]dCTP and 240mer circular ssDNA annealed with the 120mer ssDNA without (lanes 1 and 2) or with (lanes 3 and 4) abasic site were digested (lanes 2 and 4) or not (lanes 1 and 3) by Dra I and separated in a 6% polyacrylamide gel + 6 M urea along with marker DNA. Note that the 240mer circular ssDNA has a unique Dra I site. Upon Dra I cleavage of the replication products, the major 240mer fragment is formed along with other fragments that are located between the Dra I site and either the 3′ or 5′ ends of Okazaki fragments. Arrows show positions of the major 240-nt Dra I fragment and the 126-nt fragment located between the Dra I site and the 3′ end of the nascent fragment terminated by the abasic site. ( C ) DNA products were synthesized by reconstituted T4 replication complexes in the presence of [α- 32 P]dTTP and of 240mer circular ssDNA annealed with 120mer ssDNA without (lanes 1–3) or with (lanes 4–6) an abasic site, and separated in a 0.6% alkali gel.

    Techniques Used: DNA Synthesis, Labeling, Incubation, Blocking Assay, Synthesized, Marker

    5) Product Images from "Generation of outbred Ace2 knockout mice by RNA transfection of TALENs displaying colitis reminiscent pathophysiology and inflammation"

    Article Title: Generation of outbred Ace2 knockout mice by RNA transfection of TALENs displaying colitis reminiscent pathophysiology and inflammation

    Journal: Transgenic Research

    doi: 10.1007/s11248-014-9855-3

    Schematic representation of the designed TALENs and binding site in the murine ACE2 gene. a Top panel ; genomic structure of the ACE2 gene, where the 19 exons are depicted as boxes, of which non-coding sequences are in light grey and coding sequence in dark grey . Bottom panel ; architecture of the designed TALENs and their binding site in exon 2 of the Ace2 gene. Both TALEN arms contain a nuclear localization signal (NLS), a truncated 152 amino acid N-terminus, and a 63 amino acid C-terminus fused to a FokI nuclease. FokI ELD and FokI KKR represent the 3-aa substituted versions of the original FokI. A restriction site recognized by the endonuclease Dra I is underlined and in italic . b Schematic representation of the final TALEN construct pTAL-3.1 featuring a cytomegalovirus (CMV) promoter, a T7 RNA polymerase promoter (T7), either the FokI ELD or FokI KKR TALEN arm juxtaposed to a bovine growth hormone polyadenylation signal (BGH pA) and flanked by a neomycin selection marker driven by an ampicillin resistance promoter (bla) completed with a simian virus 40 polyadenylation signal (SV40 pA)
    Figure Legend Snippet: Schematic representation of the designed TALENs and binding site in the murine ACE2 gene. a Top panel ; genomic structure of the ACE2 gene, where the 19 exons are depicted as boxes, of which non-coding sequences are in light grey and coding sequence in dark grey . Bottom panel ; architecture of the designed TALENs and their binding site in exon 2 of the Ace2 gene. Both TALEN arms contain a nuclear localization signal (NLS), a truncated 152 amino acid N-terminus, and a 63 amino acid C-terminus fused to a FokI nuclease. FokI ELD and FokI KKR represent the 3-aa substituted versions of the original FokI. A restriction site recognized by the endonuclease Dra I is underlined and in italic . b Schematic representation of the final TALEN construct pTAL-3.1 featuring a cytomegalovirus (CMV) promoter, a T7 RNA polymerase promoter (T7), either the FokI ELD or FokI KKR TALEN arm juxtaposed to a bovine growth hormone polyadenylation signal (BGH pA) and flanked by a neomycin selection marker driven by an ampicillin resistance promoter (bla) completed with a simian virus 40 polyadenylation signal (SV40 pA)

    Techniques Used: TALENs, Binding Assay, Sequencing, Construct, Selection, Marker

    Identification and subdivision of TALEN-induced deletions in founder mice. a Genotyping of all newborn mice by gel electrophoresis of DraI digested PCR products spanning the Ace2 gene target site. The uncut bands ( top ) signify the mutated alleles with a deleted Dra I site. The lower two bands indicate a wt allele whereas a combination is indicative of a mosaicism. Male mice IDs are in red and female IDs in black . The encircled IDs correspond to the founder animals giving rise to the F1 population. b Animals are stratified into a male and female group, constituting 54 and 46 % of the whole population, respectively. Sequencing identified deletions are binned according to size and the percent-wise constitution of these in the two groups is depicted. c Cumulative frequency of base loss around the Ace2 target site. Deletions in male or heterologous female mice produce a count of one whereas biallelic deletions give a count of two. Mosaic animals are excluded. TALEN binding sites are shown in red. (Color figure online)
    Figure Legend Snippet: Identification and subdivision of TALEN-induced deletions in founder mice. a Genotyping of all newborn mice by gel electrophoresis of DraI digested PCR products spanning the Ace2 gene target site. The uncut bands ( top ) signify the mutated alleles with a deleted Dra I site. The lower two bands indicate a wt allele whereas a combination is indicative of a mosaicism. Male mice IDs are in red and female IDs in black . The encircled IDs correspond to the founder animals giving rise to the F1 population. b Animals are stratified into a male and female group, constituting 54 and 46 % of the whole population, respectively. Sequencing identified deletions are binned according to size and the percent-wise constitution of these in the two groups is depicted. c Cumulative frequency of base loss around the Ace2 target site. Deletions in male or heterologous female mice produce a count of one whereas biallelic deletions give a count of two. Mosaic animals are excluded. TALEN binding sites are shown in red. (Color figure online)

    Techniques Used: Mouse Assay, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Sequencing, Binding Assay

    6) Product Images from "Rapid single nucleotide polymorphism mapping in C. elegans"

    Article Title: Rapid single nucleotide polymorphism mapping in C. elegans

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-6-118

    Interval mapping . Individual recombinants are singled from heterozygotes and the animal, or a representative sample of their progeny, are placed in wells of a 96-well PCR plate and lysed. The plate may also contain three control wells, with Bristol, Hawaiian, and a 50-50 mix of animals. DNAs from the lysed animals are pin-replicated into a PCR master mix containing primers for the desired SNP. The plates are processed for PCR amplification, digested with Dra I and samples run on an agarose gel.
    Figure Legend Snippet: Interval mapping . Individual recombinants are singled from heterozygotes and the animal, or a representative sample of their progeny, are placed in wells of a 96-well PCR plate and lysed. The plate may also contain three control wells, with Bristol, Hawaiian, and a 50-50 mix of animals. DNAs from the lysed animals are pin-replicated into a PCR master mix containing primers for the desired SNP. The plates are processed for PCR amplification, digested with Dra I and samples run on an agarose gel.

    Techniques Used: Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis

    7) Product Images from "Culture time of vitrified/warmed zygotes before microinjection affects the production efficiency of CRISPR-Cas9-mediated knock-in mice"

    Article Title: Culture time of vitrified/warmed zygotes before microinjection affects the production efficiency of CRISPR-Cas9-mediated knock-in mice

    Journal: Biology Open

    doi: 10.1242/bio.025122

    Generation of knock-in mice at the Spp1 locus. (A) Schematic illustration to generate a PITChed allele at the Spp1 locus, mediated by the CRIS-PITCh (v2) system. Four glutamine residues in exons 3 and 4 (three in exon 3 and one in exon 4) were replaced with alanine residues (red stars). Two gene-specific gRNAs were designed within exon 3 and downstream of exon 4. A PITCh donor plasmid was designed to carry the substituted sequences encoding four alanine residues, silent mutations for RFLP analysis (from T to G in exon 3 and from G to A in exon 4) and a point mutation (from T to C) in intron region. Bold black arrows indicate primers for PCR. Yellow and blue arrows indicate the recognition sites of restriction enzymes for the RFLP analyses. (B) Sequence analysis of subcloned PCR products from pups harboring the knock-in allele. The sequences around exon 3 and exon 4 are displayed in the upper and lower panels, respectively. The modified codons encoding four alanines are enclosed in red boxes. The silent mutations for RFLP analyses are enclosed in green boxes. The gRNA-blocking mutation is enclosed in a purple box. The wild-type allele is shown at the top ( Spp1 Wild) with the gRNA target sequences (underlined in yellow and blue). The PAM sequences are enclosed in yellow and blue boxes. Uppercase letters indicate exon sequences. Dots indicate the same bases as the wild-type sequence. Dashes indicate deletions. Unintended mutations are enclosed in black boxes. Black underlined sequences indicate Nar I and Dra I sites in exons 3 and exon 4, respectively.
    Figure Legend Snippet: Generation of knock-in mice at the Spp1 locus. (A) Schematic illustration to generate a PITChed allele at the Spp1 locus, mediated by the CRIS-PITCh (v2) system. Four glutamine residues in exons 3 and 4 (three in exon 3 and one in exon 4) were replaced with alanine residues (red stars). Two gene-specific gRNAs were designed within exon 3 and downstream of exon 4. A PITCh donor plasmid was designed to carry the substituted sequences encoding four alanine residues, silent mutations for RFLP analysis (from T to G in exon 3 and from G to A in exon 4) and a point mutation (from T to C) in intron region. Bold black arrows indicate primers for PCR. Yellow and blue arrows indicate the recognition sites of restriction enzymes for the RFLP analyses. (B) Sequence analysis of subcloned PCR products from pups harboring the knock-in allele. The sequences around exon 3 and exon 4 are displayed in the upper and lower panels, respectively. The modified codons encoding four alanines are enclosed in red boxes. The silent mutations for RFLP analyses are enclosed in green boxes. The gRNA-blocking mutation is enclosed in a purple box. The wild-type allele is shown at the top ( Spp1 Wild) with the gRNA target sequences (underlined in yellow and blue). The PAM sequences are enclosed in yellow and blue boxes. Uppercase letters indicate exon sequences. Dots indicate the same bases as the wild-type sequence. Dashes indicate deletions. Unintended mutations are enclosed in black boxes. Black underlined sequences indicate Nar I and Dra I sites in exons 3 and exon 4, respectively.

    Techniques Used: Knock-In, Mouse Assay, Plasmid Preparation, Mutagenesis, Polymerase Chain Reaction, Sequencing, Modification, Blocking Assay

    8) Product Images from "Chromosomal Integration of a Cephalosporinase Gene from Acinetobacter baumannii into Oligella urethralis as a Source of Acquired Resistance to ?-Lactams"

    Article Title: Chromosomal Integration of a Cephalosporinase Gene from Acinetobacter baumannii into Oligella urethralis as a Source of Acquired Resistance to ?-Lactams

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.47.5.1536-1542.2003

    PFGE profiles of Sfi I-, Apa I-, Dra I-, Spe I-, and Xba I-digested whole-cell DNA of O. urethralis COH-1 (A; lanes 1 to 5) and their Southern transfer, followed by hybridization with internal probes specific for bla ABA-1 (B) and bla CARB-8 (C). Lanes M, molecular size markers
    Figure Legend Snippet: PFGE profiles of Sfi I-, Apa I-, Dra I-, Spe I-, and Xba I-digested whole-cell DNA of O. urethralis COH-1 (A; lanes 1 to 5) and their Southern transfer, followed by hybridization with internal probes specific for bla ABA-1 (B) and bla CARB-8 (C). Lanes M, molecular size markers

    Techniques Used: Hybridization

    9) Product Images from "SMN1 dosage analysis in spinal muscular atrophy from India"

    Article Title: SMN1 dosage analysis in spinal muscular atrophy from India

    Journal: BMC Medical Genetics

    doi: 10.1186/1471-2350-6-22

    SMN1 copy number analysis for the family of Case 3. 1A The pedigree of the family of Case 3. 1B: Exon 7 PCR-RFLP Polyacrylamide gels showing the undigested and digested products of SMN exon 7 after Dra I digestion. Lane 1, undigested product of patient; Lane 2, digested product of patient with SMN1 deletion; Lane 3, undigested product of sibling; Lane 4, digested product of sibling with no SMN1 deletion. 1C: Exon 8 PCR-RFLP after Dde I digestion. Lanes 1 and 3, undigested PCR product; Lane 2, digested product of patient with homozygous deletion of exon 8 of the SMN1 gene; Lane 4, digested product of sibling with no homozygous deletion of SMN1 . 1D Quantitative PCR gel scans of Father (I: 1), Mother (I: 2) and sibling (II: 4). Band sizes in base pairs are shown in the upper boxes and peak areas in the lower boxes. IS, internal standards.
    Figure Legend Snippet: SMN1 copy number analysis for the family of Case 3. 1A The pedigree of the family of Case 3. 1B: Exon 7 PCR-RFLP Polyacrylamide gels showing the undigested and digested products of SMN exon 7 after Dra I digestion. Lane 1, undigested product of patient; Lane 2, digested product of patient with SMN1 deletion; Lane 3, undigested product of sibling; Lane 4, digested product of sibling with no SMN1 deletion. 1C: Exon 8 PCR-RFLP after Dde I digestion. Lanes 1 and 3, undigested PCR product; Lane 2, digested product of patient with homozygous deletion of exon 8 of the SMN1 gene; Lane 4, digested product of sibling with no homozygous deletion of SMN1 . 1D Quantitative PCR gel scans of Father (I: 1), Mother (I: 2) and sibling (II: 4). Band sizes in base pairs are shown in the upper boxes and peak areas in the lower boxes. IS, internal standards.

    Techniques Used: Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    10) Product Images from "Heterogeneity in the vanB Gene Cluster of Genomically Diverse Clinical Strains of Vancomycin-Resistant Enterococci"

    Article Title: Heterogeneity in the vanB Gene Cluster of Genomically Diverse Clinical Strains of Vancomycin-Resistant Enterococci

    Journal: Antimicrobial Agents and Chemotherapy

    doi:

    Analysis of vanB long PCR amplicons. (Top) Representative agarose electrophoresis gel of vanB long PCR amplicons. Lanes 1 and 6, 1-kb ladder (Life Technologies, Gaithersburg, Md.); lane 2, vanB2 isolate TUH2-18; lane 3, vanB3 isolate TUH7-68; lane 4, vanB2 isolate TUH7-15 with a 789-bp insertion; lane 5, vanB1 isolate TUH4-64. (Bottom) Restriction fragment analysis of vanB long PCR amplicons. Shown are Bsp HI/ Dra I-digested vanB long PCR amplicons analyzed by agarose gel electrophoresis. Lanes 1 and 6, 1-kb ladder; lane 2, vanB2 isolate TUH2-18 with RFLP-2; lane 3, vanB3 isolate TUH7-68 RFLP-2; lane 4, vanB2 isolate TUH7-15 with a 789-bp enlargement of fragment 4 (RFLP-2*); lane 5, vanB1 isolate TUH4-64 with RFLP-1. Molecular sizes shown to the left of each gel (in base pairs) refer to the 1-kb ladder.
    Figure Legend Snippet: Analysis of vanB long PCR amplicons. (Top) Representative agarose electrophoresis gel of vanB long PCR amplicons. Lanes 1 and 6, 1-kb ladder (Life Technologies, Gaithersburg, Md.); lane 2, vanB2 isolate TUH2-18; lane 3, vanB3 isolate TUH7-68; lane 4, vanB2 isolate TUH7-15 with a 789-bp insertion; lane 5, vanB1 isolate TUH4-64. (Bottom) Restriction fragment analysis of vanB long PCR amplicons. Shown are Bsp HI/ Dra I-digested vanB long PCR amplicons analyzed by agarose gel electrophoresis. Lanes 1 and 6, 1-kb ladder; lane 2, vanB2 isolate TUH2-18 with RFLP-2; lane 3, vanB3 isolate TUH7-68 RFLP-2; lane 4, vanB2 isolate TUH7-15 with a 789-bp enlargement of fragment 4 (RFLP-2*); lane 5, vanB1 isolate TUH4-64 with RFLP-1. Molecular sizes shown to the left of each gel (in base pairs) refer to the 1-kb ladder.

    Techniques Used: Polymerase Chain Reaction, Electrophoresis, Agarose Gel Electrophoresis

    11) Product Images from "Atypical 16S rRNA Gene Copies in Ochrobactrum intermedium Strains Reveal a Large Genomic Rearrangement by Recombination between rrn Copies"

    Article Title: Atypical 16S rRNA Gene Copies in Ochrobactrum intermedium Strains Reveal a Large Genomic Rearrangement by Recombination between rrn Copies

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.185.9.2901-2909.2003

    Expression of the 46-bp insertion and detection of the two types of 16S rRNA by RT-PCR. The strains analyzed are indicated at the top. (A) Lanes +, use of consensual primer pair 27f and 590r; lanes ins, use of an insertion-specific pair of primers, ins1 and 590r. (B) Lanes +, RT-PCR products obtained with the primers 27f and 590r; lanes Dra I, RT-PCR products obtained in + lanes digested by Dra I. Lanes − (A and B), negative controls performed on the RNA of strain PR17/sat without reverse transcription and with the primers 27f and 590r (negative controls were also obtained for other strains and for the primers ins1 and 590r but are not shown); lane M, 100-bp DNA ladder (Promega) as a molecular weight marker.
    Figure Legend Snippet: Expression of the 46-bp insertion and detection of the two types of 16S rRNA by RT-PCR. The strains analyzed are indicated at the top. (A) Lanes +, use of consensual primer pair 27f and 590r; lanes ins, use of an insertion-specific pair of primers, ins1 and 590r. (B) Lanes +, RT-PCR products obtained with the primers 27f and 590r; lanes Dra I, RT-PCR products obtained in + lanes digested by Dra I. Lanes − (A and B), negative controls performed on the RNA of strain PR17/sat without reverse transcription and with the primers 27f and 590r (negative controls were also obtained for other strains and for the primers ins1 and 590r but are not shown); lane M, 100-bp DNA ladder (Promega) as a molecular weight marker.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Molecular Weight, Marker

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    Article Title: Circular RNA biogenesis can proceed through an exon-containing lariat precursor
    Article Snippet: .. 20 U of Dra I (NEB) was added directly to the 50 μl PCR reaction, and the reaction was allowed to incubate at 37°C for 1 hr. .. From there, the PCR was resumed for 35 additional cycles without purification. qPCR reactions were assembled as 10 μl reactions using AccuPower 2X GreenStar qPCR Master Mix (Bioneer) and Power SYBR Green Master Mix (Life Technologies) with 0.3 μl of template used per reaction.

    Article Title: Promoterless Transposon Mutagenesis Drives Solid Cancers via Tumor Suppressor Inactivation
    Article Snippet: Generation of T2/Onc2.3 transgenic mice pT2/Onc2.3 DNA was linearized with restriction enzyme with Sca I (NEB #R3122) at position 3,502 bp ( ) at 37°C for 90 m, followed by heat inactivation at 80 °C for 20 m. pT2/Onc2.3:Sca I linear plasmid, at 2 ng/uL, 4 ng/uL and 10 ng/uL, was prepared for microinjection into (B6C3)F2 hybrid embryos using standard techniques ( ). .. Tail biopsy genomic DNA from founder animals was digested with Dra I (NEB) and Bam HI (NEB), run on a 0.8% TAE agarose gel at 30 v for 16 h, transferred to membrane, and screened by Southern blotting using a 32 P-labeled En2-SA (splice acceptor) probe, a 500 bp En2-SA PCR product with pT2/Onc2.3 template and primers T2.3-En2.5Probe, 5’-GCTGCAATAAACAAGTTGGCCG-3’ and T2.3-En2.3Probe, 5’-CTTGGGTCAAACATTTCGAGTAGCC-3’, and standard methods. .. Individual transgenic lines (n=5) were established by backcrossing founders to C57BL/6J (JAX#000664).

    Article Title: Rescuing ocular development in an anophthalmic pig by blastocyst complementation
    Article Snippet: DNA samples were analyzed using PCR with specific primers for the MITF gene and restriction fragment length polymorphism (RFLP) analysis. .. Eight microliters of PCR product was digested with DraI (New England Biolabs, USA). .. The full‐length reaction products were 471 bp and were separated into 261 and 210 bp by 2% agarose gel electrophoresis in the presence of ethidium bromide solution and visualized with a UV transilluminator (UVP, Upland, CA, USA).

    Agarose Gel Electrophoresis:

    Article Title: Promoterless Transposon Mutagenesis Drives Solid Cancers via Tumor Suppressor Inactivation
    Article Snippet: Generation of T2/Onc2.3 transgenic mice pT2/Onc2.3 DNA was linearized with restriction enzyme with Sca I (NEB #R3122) at position 3,502 bp ( ) at 37°C for 90 m, followed by heat inactivation at 80 °C for 20 m. pT2/Onc2.3:Sca I linear plasmid, at 2 ng/uL, 4 ng/uL and 10 ng/uL, was prepared for microinjection into (B6C3)F2 hybrid embryos using standard techniques ( ). .. Tail biopsy genomic DNA from founder animals was digested with Dra I (NEB) and Bam HI (NEB), run on a 0.8% TAE agarose gel at 30 v for 16 h, transferred to membrane, and screened by Southern blotting using a 32 P-labeled En2-SA (splice acceptor) probe, a 500 bp En2-SA PCR product with pT2/Onc2.3 template and primers T2.3-En2.5Probe, 5’-GCTGCAATAAACAAGTTGGCCG-3’ and T2.3-En2.3Probe, 5’-CTTGGGTCAAACATTTCGAGTAGCC-3’, and standard methods. .. Individual transgenic lines (n=5) were established by backcrossing founders to C57BL/6J (JAX#000664).

    Southern Blot:

    Article Title: Promoterless Transposon Mutagenesis Drives Solid Cancers via Tumor Suppressor Inactivation
    Article Snippet: Generation of T2/Onc2.3 transgenic mice pT2/Onc2.3 DNA was linearized with restriction enzyme with Sca I (NEB #R3122) at position 3,502 bp ( ) at 37°C for 90 m, followed by heat inactivation at 80 °C for 20 m. pT2/Onc2.3:Sca I linear plasmid, at 2 ng/uL, 4 ng/uL and 10 ng/uL, was prepared for microinjection into (B6C3)F2 hybrid embryos using standard techniques ( ). .. Tail biopsy genomic DNA from founder animals was digested with Dra I (NEB) and Bam HI (NEB), run on a 0.8% TAE agarose gel at 30 v for 16 h, transferred to membrane, and screened by Southern blotting using a 32 P-labeled En2-SA (splice acceptor) probe, a 500 bp En2-SA PCR product with pT2/Onc2.3 template and primers T2.3-En2.5Probe, 5’-GCTGCAATAAACAAGTTGGCCG-3’ and T2.3-En2.3Probe, 5’-CTTGGGTCAAACATTTCGAGTAGCC-3’, and standard methods. .. Individual transgenic lines (n=5) were established by backcrossing founders to C57BL/6J (JAX#000664).

    Incubation:

    Article Title: Biochemical characterization of a cancer-associated E109K missense variant of human exonuclease 1
    Article Snippet: Briefly, the heteroduplex phagemid DNA substrate (48 fmol) containing a T/G mismatch in its unique SalI site and a single nick generated by Nt·BstNBI 361 nucleotides 5′ from the mispaired T was incubated with 100 μg of nuclear extracts of HEK293 cells pretreated or not with EXO1 siRNA and supplemented with 40 nM Exo1 wt, E109K or D173A in 20 mM Tris·HCl pH 7.6, 110 mM KCl, 5 mM MgCl2 , 1 mM glutathione, 1.5 mM ATP, 50 μg/ml BSA and 100 μM dNTPs for 30 min in a total volume of 25 μl. .. The reactions were terminated by a 30-min incubation with a stop solution (final concentrations: 0.5 mM EDTA, 1.5% SDS (sodium dodecyl sulfate), 2.5 mg/ml proteinase K), cleaned up on a MinElute column (Qiagen), and the recovered phagemid was subjected to restriction digest with 6 U SalI and 20 U DraI (NEB). .. RNase A (40 ng, Sigma-Aldrich) was then added and, following an overnight incubation at 37°C, the reaction products were separated on a 1% agarose gel eluted with TAE buffer and stained with GelRed.

    Article Title: Analysis of Distant Communication on Defined Chromatin Templates In Vitro
    Article Snippet: However a similar technique could be applied to any DNA region on the plasmid ( , ). .. 750 ng of DNA or nucleosomal templates (20 μg/ml) were incubated in the presence of an excess of Dra I and Bgl II restriction endonucleases (10 units each) in the NEBuffer #2 at 37°C for 2 hours. .. DNA was purified by phenol/chloroform extraction followed by ethanol precipitation and analyzed by electrophoresis in 1% agarose/TAE gel.

    Modification:

    Article Title: Involvement of DNA ligase III and ribonuclease H1 in mitochondrial DNA replication in cultured human cells
    Article Snippet: Quantification of mtDNA was conducted using a rt-qPCR method essentially as described . .. 2.4 DNA modification, 2D-AGE and Southern hybridisation Total DNA was digested with DraI (New England Biolabs), and then precipitated and suspended in 10 mM Hepes-NaOH (pH 7.2). .. In some cases the digested DNA was further modified with 9.5 unit S1 nuclease (Promega) in 30 μl reaction mixture at 37 °C for 20 min and then 1.5 μl of 0.5 M EDTA (pH 8) was added to the reaction.

    Hybridization:

    Article Title: Involvement of DNA ligase III and ribonuclease H1 in mitochondrial DNA replication in cultured human cells
    Article Snippet: Quantification of mtDNA was conducted using a rt-qPCR method essentially as described . .. 2.4 DNA modification, 2D-AGE and Southern hybridisation Total DNA was digested with DraI (New England Biolabs), and then precipitated and suspended in 10 mM Hepes-NaOH (pH 7.2). .. In some cases the digested DNA was further modified with 9.5 unit S1 nuclease (Promega) in 30 μl reaction mixture at 37 °C for 20 min and then 1.5 μl of 0.5 M EDTA (pH 8) was added to the reaction.

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    New England Biolabs dra i
    Transgenic SB-T2/Onc2.3 founder mice. ( a ) Plasmid map of pT2/Onc2.3-MBM102, denoting essential features and base pair position in parenthesis, used to create the T2/Onc2.3 high-copy transgenic lines. ( b ) Southern blot of T2/Onc2.3 founder mice using a probe corresponding to the En2 -SA ( Engrailed 2 splice acceptor) element within the SB-T2/SB-GT allele construct that also cross hybridizes to the endogenous En2 mouse gene on chromosome 5. Genomic <t>DNA</t> digested to completion with <t>Dra</t> I restriction enzyme and detected with a radioisotope- 32 P labeled En2 -SA probe identifies the diploid endogenous En2 locus as a discrete 2.1 kb band and any all copies of the T2/SB-GT monomer that have been liberated from the their randomly inserted transgenic loci as a discrete 1.0 kb band. Mice with multi-copy concatemer alleles are identified as containing darker staining bands relative to the two copies of the En2 endogenous locus band. Blue arrows and dotted boxes denote animals selected for germ line breeding — five separate transgenic founders, three females (TG.14885, TG.14922, and TG.14942) and two males (TG.14913 and TG.14927), were selected for mating with wildtype C57BL/6J mice to confirm the germ line transmission of the SB-T/2SB-GT concatemer alleles to progeny. Mice denoted with blue dotted boxes produced transgenic carrier pups with a transgene copy numbers that differed between individuals, suggesting the presence of two or more independent transgenic concatemer integration sites. Mice denoted with red dotted boxes produced transgenic carrier pups with a transgene copy number that did not differ between individuals, suggesting that they resulted from a single transgenic concatemer integration site. Lines established from founders TG.14913, TG.14922, and TG.14942 were expanded and bred for further experiments. Bam HI restriction digested lambda (ƛ)-phage DNA is provided as a reference, a small amount was added to the radioisotope- 32 P labeling reaction with the En2 -SA probe to show detection on the developed Southern blot.
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    Transgenic SB-T2/Onc2.3 founder mice. ( a ) Plasmid map of pT2/Onc2.3-MBM102, denoting essential features and base pair position in parenthesis, used to create the T2/Onc2.3 high-copy transgenic lines. ( b ) Southern blot of T2/Onc2.3 founder mice using a probe corresponding to the En2 -SA ( Engrailed 2 splice acceptor) element within the SB-T2/SB-GT allele construct that also cross hybridizes to the endogenous En2 mouse gene on chromosome 5. Genomic DNA digested to completion with Dra I restriction enzyme and detected with a radioisotope- 32 P labeled En2 -SA probe identifies the diploid endogenous En2 locus as a discrete 2.1 kb band and any all copies of the T2/SB-GT monomer that have been liberated from the their randomly inserted transgenic loci as a discrete 1.0 kb band. Mice with multi-copy concatemer alleles are identified as containing darker staining bands relative to the two copies of the En2 endogenous locus band. Blue arrows and dotted boxes denote animals selected for germ line breeding — five separate transgenic founders, three females (TG.14885, TG.14922, and TG.14942) and two males (TG.14913 and TG.14927), were selected for mating with wildtype C57BL/6J mice to confirm the germ line transmission of the SB-T/2SB-GT concatemer alleles to progeny. Mice denoted with blue dotted boxes produced transgenic carrier pups with a transgene copy numbers that differed between individuals, suggesting the presence of two or more independent transgenic concatemer integration sites. Mice denoted with red dotted boxes produced transgenic carrier pups with a transgene copy number that did not differ between individuals, suggesting that they resulted from a single transgenic concatemer integration site. Lines established from founders TG.14913, TG.14922, and TG.14942 were expanded and bred for further experiments. Bam HI restriction digested lambda (ƛ)-phage DNA is provided as a reference, a small amount was added to the radioisotope- 32 P labeling reaction with the En2 -SA probe to show detection on the developed Southern blot.

    Journal: bioRxiv

    Article Title: Promoterless Transposon Mutagenesis Drives Solid Cancers via Tumor Suppressor Inactivation

    doi: 10.1101/2020.08.17.254565

    Figure Lengend Snippet: Transgenic SB-T2/Onc2.3 founder mice. ( a ) Plasmid map of pT2/Onc2.3-MBM102, denoting essential features and base pair position in parenthesis, used to create the T2/Onc2.3 high-copy transgenic lines. ( b ) Southern blot of T2/Onc2.3 founder mice using a probe corresponding to the En2 -SA ( Engrailed 2 splice acceptor) element within the SB-T2/SB-GT allele construct that also cross hybridizes to the endogenous En2 mouse gene on chromosome 5. Genomic DNA digested to completion with Dra I restriction enzyme and detected with a radioisotope- 32 P labeled En2 -SA probe identifies the diploid endogenous En2 locus as a discrete 2.1 kb band and any all copies of the T2/SB-GT monomer that have been liberated from the their randomly inserted transgenic loci as a discrete 1.0 kb band. Mice with multi-copy concatemer alleles are identified as containing darker staining bands relative to the two copies of the En2 endogenous locus band. Blue arrows and dotted boxes denote animals selected for germ line breeding — five separate transgenic founders, three females (TG.14885, TG.14922, and TG.14942) and two males (TG.14913 and TG.14927), were selected for mating with wildtype C57BL/6J mice to confirm the germ line transmission of the SB-T/2SB-GT concatemer alleles to progeny. Mice denoted with blue dotted boxes produced transgenic carrier pups with a transgene copy numbers that differed between individuals, suggesting the presence of two or more independent transgenic concatemer integration sites. Mice denoted with red dotted boxes produced transgenic carrier pups with a transgene copy number that did not differ between individuals, suggesting that they resulted from a single transgenic concatemer integration site. Lines established from founders TG.14913, TG.14922, and TG.14942 were expanded and bred for further experiments. Bam HI restriction digested lambda (ƛ)-phage DNA is provided as a reference, a small amount was added to the radioisotope- 32 P labeling reaction with the En2 -SA probe to show detection on the developed Southern blot.

    Article Snippet: Tail biopsy genomic DNA from founder animals was digested with Dra I (NEB) and Bam HI (NEB), run on a 0.8% TAE agarose gel at 30 v for 16 h, transferred to membrane, and screened by Southern blotting using a 32 P-labeled En2-SA (splice acceptor) probe, a 500 bp En2-SA PCR product with pT2/Onc2.3 template and primers T2.3-En2.5Probe, 5’-GCTGCAATAAACAAGTTGGCCG-3’ and T2.3-En2.3Probe, 5’-CTTGGGTCAAACATTTCGAGTAGCC-3’, and standard methods.

    Techniques: Transgenic Assay, Mouse Assay, Plasmid Preparation, Southern Blot, Construct, Labeling, Staining, Transmission Assay, Produced

    Estimating SB transposon concatemer copy number from different SB strains. ( a ) Inverse image of EtBr stained agarose gel (1.2% TBE, run at 30 volts for 30 hours) containing genomic DNA digested with Dra I for 16 hours at 37 °C for Southern blot analysis. Molecular weight markers: left, lambda cut with Hin DIII; right, 100 bp ladder. ( b ) Southern blot demonstrating relative concatemer copy number differences between high-copy and low-copy SB transposon alleles in homozygous mice with unmobilized transposons. The En2 site provides a reference for a single copy, diploid gene.

    Journal: bioRxiv

    Article Title: Promoterless Transposon Mutagenesis Drives Solid Cancers via Tumor Suppressor Inactivation

    doi: 10.1101/2020.08.17.254565

    Figure Lengend Snippet: Estimating SB transposon concatemer copy number from different SB strains. ( a ) Inverse image of EtBr stained agarose gel (1.2% TBE, run at 30 volts for 30 hours) containing genomic DNA digested with Dra I for 16 hours at 37 °C for Southern blot analysis. Molecular weight markers: left, lambda cut with Hin DIII; right, 100 bp ladder. ( b ) Southern blot demonstrating relative concatemer copy number differences between high-copy and low-copy SB transposon alleles in homozygous mice with unmobilized transposons. The En2 site provides a reference for a single copy, diploid gene.

    Article Snippet: Tail biopsy genomic DNA from founder animals was digested with Dra I (NEB) and Bam HI (NEB), run on a 0.8% TAE agarose gel at 30 v for 16 h, transferred to membrane, and screened by Southern blotting using a 32 P-labeled En2-SA (splice acceptor) probe, a 500 bp En2-SA PCR product with pT2/Onc2.3 template and primers T2.3-En2.5Probe, 5’-GCTGCAATAAACAAGTTGGCCG-3’ and T2.3-En2.3Probe, 5’-CTTGGGTCAAACATTTCGAGTAGCC-3’, and standard methods.

    Techniques: Staining, Agarose Gel Electrophoresis, Southern Blot, Molecular Weight, Mouse Assay

    Characterization of chromatin templates using restriction enzyme sensitivity assay. Chromatin was assembled on supercoiled pYP05 plasmid and digested with an excess of restriction enzymes Dra I and Bgl II. Then DNA was purified and analyzed in 1% agarose

    Journal: Methods in molecular biology (Clifton, N.J.)

    Article Title: Analysis of Distant Communication on Defined Chromatin Templates In Vitro

    doi: 10.1007/978-1-60327-015-1_33

    Figure Lengend Snippet: Characterization of chromatin templates using restriction enzyme sensitivity assay. Chromatin was assembled on supercoiled pYP05 plasmid and digested with an excess of restriction enzymes Dra I and Bgl II. Then DNA was purified and analyzed in 1% agarose

    Article Snippet: 750 ng of DNA or nucleosomal templates (20 μg/ml) were incubated in the presence of an excess of Dra I and Bgl II restriction endonucleases (10 units each) in the NEBuffer #2 at 37°C for 2 hours.

    Techniques: Sensitive Assay, Plasmid Preparation, Purification

    Additional Dra I restriction digest PCR for Y-shaped intermediate product. Panel 2D with additional lanes indicating attempted amplification of the Y-shaped intermediate. As in 2D, primers used are indicated above the gel and observed products are illustrated below. The off-target product is the higher molecular weight species (lane 13) and represents mispriming of the reverse primer to a downstream site in exon 3. DOI: http://dx.doi.org/10.7554/eLife.07540.010

    Journal: eLife

    Article Title: Circular RNA biogenesis can proceed through an exon-containing lariat precursor

    doi: 10.7554/eLife.07540

    Figure Lengend Snippet: Additional Dra I restriction digest PCR for Y-shaped intermediate product. Panel 2D with additional lanes indicating attempted amplification of the Y-shaped intermediate. As in 2D, primers used are indicated above the gel and observed products are illustrated below. The off-target product is the higher molecular weight species (lane 13) and represents mispriming of the reverse primer to a downstream site in exon 3. DOI: http://dx.doi.org/10.7554/eLife.07540.010

    Article Snippet: 20 U of Dra I (NEB) was added directly to the 50 μl PCR reaction, and the reaction was allowed to incubate at 37°C for 1 hr.

    Techniques: Polymerase Chain Reaction, Amplification, Molecular Weight

    Effect of an abasic site on DNA synthesis by reconstituted phage T4 replication complexes. ( A ) An abasic site blocks DNA synthesis by T4 DNA polymerase holoenzyme. 32 P-end-labeled 21mer #7 annealed with 120mer #3 (lanes 1, 3, 5, 7, 9, 11, 13 and 15) or with the same oligomer containing an abasic site at position 99 (120mer #4) (lanes 2, 4, 6, 8, 10, 12, 14 and 16) was incubated with either T4 gp43 (lanes 5, 6, 13 and 14) or the exonuclease-deficient gp43D219A (lanes 7, 8, 15 and 16) in the presence of gp44/62, gp45 and gp32 for the indicated times, and the products were separated in a 10% polyacrylamide gel + 6 M urea. Note that in the substrate with an abasic site, the first nucleotide to be incorporated into the 32 P-end-labeled 21mer is opposite the abasic site. ( B ) Block to lagging-strand synthesis imposed by the abasic site. Products of DNA synthesized by reconstituted T4 replication complexes in the presence of [α- 32 P]dCTP and 240mer circular ssDNA annealed with the 120mer ssDNA without (lanes 1 and 2) or with (lanes 3 and 4) abasic site were digested (lanes 2 and 4) or not (lanes 1 and 3) by Dra I and separated in a 6% polyacrylamide gel + 6 M urea along with marker DNA. Note that the 240mer circular ssDNA has a unique Dra I site. Upon Dra I cleavage of the replication products, the major 240mer fragment is formed along with other fragments that are located between the Dra I site and either the 3′ or 5′ ends of Okazaki fragments. Arrows show positions of the major 240-nt Dra I fragment and the 126-nt fragment located between the Dra I site and the 3′ end of the nascent fragment terminated by the abasic site. ( C ) DNA products were synthesized by reconstituted T4 replication complexes in the presence of [α- 32 P]dTTP and of 240mer circular ssDNA annealed with 120mer ssDNA without (lanes 1–3) or with (lanes 4–6) an abasic site, and separated in a 0.6% alkali gel.

    Journal: Nucleic Acids Research

    Article Title: Characterization of DNA synthesis catalyzed by bacteriophage T4 replication complexes reconstituted on synthetic circular substrates

    doi:

    Figure Lengend Snippet: Effect of an abasic site on DNA synthesis by reconstituted phage T4 replication complexes. ( A ) An abasic site blocks DNA synthesis by T4 DNA polymerase holoenzyme. 32 P-end-labeled 21mer #7 annealed with 120mer #3 (lanes 1, 3, 5, 7, 9, 11, 13 and 15) or with the same oligomer containing an abasic site at position 99 (120mer #4) (lanes 2, 4, 6, 8, 10, 12, 14 and 16) was incubated with either T4 gp43 (lanes 5, 6, 13 and 14) or the exonuclease-deficient gp43D219A (lanes 7, 8, 15 and 16) in the presence of gp44/62, gp45 and gp32 for the indicated times, and the products were separated in a 10% polyacrylamide gel + 6 M urea. Note that in the substrate with an abasic site, the first nucleotide to be incorporated into the 32 P-end-labeled 21mer is opposite the abasic site. ( B ) Block to lagging-strand synthesis imposed by the abasic site. Products of DNA synthesized by reconstituted T4 replication complexes in the presence of [α- 32 P]dCTP and 240mer circular ssDNA annealed with the 120mer ssDNA without (lanes 1 and 2) or with (lanes 3 and 4) abasic site were digested (lanes 2 and 4) or not (lanes 1 and 3) by Dra I and separated in a 6% polyacrylamide gel + 6 M urea along with marker DNA. Note that the 240mer circular ssDNA has a unique Dra I site. Upon Dra I cleavage of the replication products, the major 240mer fragment is formed along with other fragments that are located between the Dra I site and either the 3′ or 5′ ends of Okazaki fragments. Arrows show positions of the major 240-nt Dra I fragment and the 126-nt fragment located between the Dra I site and the 3′ end of the nascent fragment terminated by the abasic site. ( C ) DNA products were synthesized by reconstituted T4 replication complexes in the presence of [α- 32 P]dTTP and of 240mer circular ssDNA annealed with 120mer ssDNA without (lanes 1–3) or with (lanes 4–6) an abasic site, and separated in a 0.6% alkali gel.

    Article Snippet: T4 DNA ligase, Dra I and polynucleotide kinase were from New England Biolabs.

    Techniques: DNA Synthesis, Labeling, Incubation, Blocking Assay, Synthesized, Marker