phusion dna polymerase  (New England Biolabs)


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    Name:
    Phusion High Fidelity DNA Polymerase
    Description:
    Phusion High Fidelity DNA Polymerase 500 units
    Catalog Number:
    m0530l
    Price:
    446
    Size:
    500 units
    Category:
    Thermostable DNA Polymerases
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    Structured Review

    New England Biolabs phusion dna polymerase
    Phusion High Fidelity DNA Polymerase
    Phusion High Fidelity DNA Polymerase 500 units
    https://www.bioz.com/result/phusion dna polymerase/product/New England Biolabs
    Average 99 stars, based on 726 article reviews
    Price from $9.99 to $1999.99
    phusion dna polymerase - by Bioz Stars, 2020-08
    99/100 stars

    Images

    1) Product Images from "Enzymatic Synthesis of Modified Oligonucleotides by PEAR Using Phusion and KOD DNA Polymerases"

    Article Title: Enzymatic Synthesis of Modified Oligonucleotides by PEAR Using Phusion and KOD DNA Polymerases

    Journal: Nucleic Acid Therapeutics

    doi: 10.1089/nat.2014.0513

    Polyacrylamide gel electrophoresis (PAGE) electrophoresis of the polymerase–endonuclease amplification reaction (PEAR) products. Lowercase letters (agct) represents unmodified dNTPs; uppercase letters (AGCT) represent modified dNTPs (2′-F-dNTPs or dNTPαSs). (A) PEAR by Phusion DNA polymerase using unmodified dNTPs, 2′-F-modified dATP and dGTP, respectively. Lane 1: 10-bp DNA ladder; lane 2: normal dNTPs; lane 3: 2′-F-dATP modified PEAR products; lane 4: control without PspGI; lane 5: control without Phusion DNA polymerase; lane 6: control without dATP; lane 7: 10-bp DNA ladder; lane 8: 2′-F-dGTP modified PEAR products; lane 9: control without PspGI; lane 10 : control without Phusion DNA polymerase; lane 11: control without dGTP. (B) PEAR by Phusion DNA polymerase using 2′-F-dCTP and 2′-F-dUTP. Lane 1: 2′-F-dCTP modified PEAR products; lane 2: control without PspGI; lane 3: control without Phusion DNA polymerase; lane 4: control without dCTP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dUTP modified PEAR products; lane 7: control without PspGI; lane 8: control without Phusion DNA polymerase; lane 9: control without dUTP; lane 10: 10-bp DNA ladder. (C) 2′-F-dATP and 2′-F-dGTP modified PEAR products as “seeds” for PEAR. Lane 1: 10-bp DNA ladder; lane 2: control without PspGI; lane 3: using 2′-F-dATP modified PEAR products as “seeds” for PEAR; lane 4: control without PspGI; lane 5: using 2′-F-dGTP modified PEAR products as seeds for PEAR. (D) 2′-F-dATP and 2′-F-dGTP modified PEAR products using KOD DNA polymerase. Lane 1: 2′-F-dATP modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without 2′-F-dATP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dGTP modified PEAR products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase ; lane 9: control without 2′-F-dGTP; lane 10: 10-bp DNA ladder. (E) PEAR amplification of 2′-F-dCTP and 2′-F-dUTP modified products using KOD DNA polymerase. Lane 1: 2′-F-dCTP modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without dCTP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dUTP modified PEAR products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without dUTP; lane 10: 10-bp DNA ladder. (F) PEAR amplification of dTTPαS modified and 2′-F-dATP+dGTPαS double modified PEAR products using KOD DNA polymerase. Lane 1: dTTPαS modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without dTTPαS; lane 5: 20 bp DNA ladder; lane 6: 2′-F-dATP and dGTPαS double modified PEAR amplified products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without 2′-F-dATP and dGTPαS; lane 10: 20-bp DNA ladder. (G) PEAR amplification of 2′-F-dATP+dCTPαS double modified and 2′-F-dATP+dTTPαS double modified PEAR products using KOD DNA polymerase. Lane 1: 2′-F-dATP+dCTPαS double modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without 2′-F-dATP and dCTPαS; lane 5: 20-bp DNA ladder; lane 6: 2′-F-dATP+dTTPαS double modified PEAR amplified products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without 2′-F-dGTP and dTTPαS; lane 10: 20-bp DNA ladder.
    Figure Legend Snippet: Polyacrylamide gel electrophoresis (PAGE) electrophoresis of the polymerase–endonuclease amplification reaction (PEAR) products. Lowercase letters (agct) represents unmodified dNTPs; uppercase letters (AGCT) represent modified dNTPs (2′-F-dNTPs or dNTPαSs). (A) PEAR by Phusion DNA polymerase using unmodified dNTPs, 2′-F-modified dATP and dGTP, respectively. Lane 1: 10-bp DNA ladder; lane 2: normal dNTPs; lane 3: 2′-F-dATP modified PEAR products; lane 4: control without PspGI; lane 5: control without Phusion DNA polymerase; lane 6: control without dATP; lane 7: 10-bp DNA ladder; lane 8: 2′-F-dGTP modified PEAR products; lane 9: control without PspGI; lane 10 : control without Phusion DNA polymerase; lane 11: control without dGTP. (B) PEAR by Phusion DNA polymerase using 2′-F-dCTP and 2′-F-dUTP. Lane 1: 2′-F-dCTP modified PEAR products; lane 2: control without PspGI; lane 3: control without Phusion DNA polymerase; lane 4: control without dCTP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dUTP modified PEAR products; lane 7: control without PspGI; lane 8: control without Phusion DNA polymerase; lane 9: control without dUTP; lane 10: 10-bp DNA ladder. (C) 2′-F-dATP and 2′-F-dGTP modified PEAR products as “seeds” for PEAR. Lane 1: 10-bp DNA ladder; lane 2: control without PspGI; lane 3: using 2′-F-dATP modified PEAR products as “seeds” for PEAR; lane 4: control without PspGI; lane 5: using 2′-F-dGTP modified PEAR products as seeds for PEAR. (D) 2′-F-dATP and 2′-F-dGTP modified PEAR products using KOD DNA polymerase. Lane 1: 2′-F-dATP modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without 2′-F-dATP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dGTP modified PEAR products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase ; lane 9: control without 2′-F-dGTP; lane 10: 10-bp DNA ladder. (E) PEAR amplification of 2′-F-dCTP and 2′-F-dUTP modified products using KOD DNA polymerase. Lane 1: 2′-F-dCTP modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without dCTP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dUTP modified PEAR products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without dUTP; lane 10: 10-bp DNA ladder. (F) PEAR amplification of dTTPαS modified and 2′-F-dATP+dGTPαS double modified PEAR products using KOD DNA polymerase. Lane 1: dTTPαS modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without dTTPαS; lane 5: 20 bp DNA ladder; lane 6: 2′-F-dATP and dGTPαS double modified PEAR amplified products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without 2′-F-dATP and dGTPαS; lane 10: 20-bp DNA ladder. (G) PEAR amplification of 2′-F-dATP+dCTPαS double modified and 2′-F-dATP+dTTPαS double modified PEAR products using KOD DNA polymerase. Lane 1: 2′-F-dATP+dCTPαS double modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without 2′-F-dATP and dCTPαS; lane 5: 20-bp DNA ladder; lane 6: 2′-F-dATP+dTTPαS double modified PEAR amplified products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without 2′-F-dGTP and dTTPαS; lane 10: 20-bp DNA ladder.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Electrophoresis, Amplification, Modification

    2) Product Images from "Variations of five eIF4E genes across cassava accessions exhibiting tolerant and susceptible responses to cassava brown streak disease"

    Article Title: Variations of five eIF4E genes across cassava accessions exhibiting tolerant and susceptible responses to cassava brown streak disease

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0181998

    RT-PCR amplification of five cassava eIF4E ORFs with gene-specific primers. Total RNA was extracted from TMS60444 cassava line. The first-strand cDNA was synthesized using SuperScript ™ III Reverse Transcriptase (Invitrogen) and PCR was performed using Phusion High-Fidelity DNA polymerase (New England Biolabs) and primers indicated in Table 1 . Lane 1: DNA marker; Lane 2: 016601 CDS amplified with primers 016601F and 016601R; Lane 3: 016620 CDS amplified with primers 016620F and 016620R; Lane 4: 015501 CDS amplified with 015501F and 015501R, Lane 5: 013223 CDS amplified with primers 013223lF and 013223R, Lane 6, 013223 CDS amplified with primers 013223sF and 013223R; Lane 7: 013732 CDS amplified with 013732lF and 013732R; Lane 8: 013732 CDS amplified with 013732sF and 013732R; and Lane 9: negative water control.
    Figure Legend Snippet: RT-PCR amplification of five cassava eIF4E ORFs with gene-specific primers. Total RNA was extracted from TMS60444 cassava line. The first-strand cDNA was synthesized using SuperScript ™ III Reverse Transcriptase (Invitrogen) and PCR was performed using Phusion High-Fidelity DNA polymerase (New England Biolabs) and primers indicated in Table 1 . Lane 1: DNA marker; Lane 2: 016601 CDS amplified with primers 016601F and 016601R; Lane 3: 016620 CDS amplified with primers 016620F and 016620R; Lane 4: 015501 CDS amplified with 015501F and 015501R, Lane 5: 013223 CDS amplified with primers 013223lF and 013223R, Lane 6, 013223 CDS amplified with primers 013223sF and 013223R; Lane 7: 013732 CDS amplified with 013732lF and 013732R; Lane 8: 013732 CDS amplified with 013732sF and 013732R; and Lane 9: negative water control.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Synthesized, Polymerase Chain Reaction, Marker

    3) Product Images from "Deep Sequencing for Evaluation of Genetic Stability of Influenza A/California/07/2009 (H1N1) Vaccine Viruses"

    Article Title: Deep Sequencing for Evaluation of Genetic Stability of Influenza A/California/07/2009 (H1N1) Vaccine Viruses

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0138650

    A: Simultaneous RT-PCR amplification of the eight segments of A/California/07/2009 (H1N1) vaccine viruses using Phusion DNA polymerase, 1: 1 kb DNA Ladder, 2: X-181-M1, 3: X-179A-M1, 4: X-181-M2, 5: X-179A-M2, 6: X-181-M3, 7: X-179A-M4, 8: 1 kb DNA Ladder, 9: 1 kb DNA Ladder, 10: 121XP-M4, 11: X-181-M4, 12: X-179A-M3, 13: X-179A-M5, 14: X-179A, 15: X-181, 16: Negative control (H 2 O). B: Simultaneous RT-PCR amplification of the eight segments of A/California/07/2009 (H1N1) vaccine viruses using Thermo Scientist Extensor Mix, 1: 1 kb DNA Ladder, 2: X-181-M1, 3: X-179A-M1, 4: X-181-M2, 5: X-179A-M2, 6: X-181-M3, 7: X-179A-M4, 8: Negative control (H 2 O), 9: 1 kb DNA Ladder, 10: 121XP-M4, 11: X-181-M4, 12: X-179A-M3, 13: X-179A-M5, 14: X-179A, 15: X-181, 16: Negative control (H 2 O). C: The distribution amount of sequencing reads between the eight segments of influenza virus using different approaches of library preparation followed by Illumina HiSeq sequencing.
    Figure Legend Snippet: A: Simultaneous RT-PCR amplification of the eight segments of A/California/07/2009 (H1N1) vaccine viruses using Phusion DNA polymerase, 1: 1 kb DNA Ladder, 2: X-181-M1, 3: X-179A-M1, 4: X-181-M2, 5: X-179A-M2, 6: X-181-M3, 7: X-179A-M4, 8: 1 kb DNA Ladder, 9: 1 kb DNA Ladder, 10: 121XP-M4, 11: X-181-M4, 12: X-179A-M3, 13: X-179A-M5, 14: X-179A, 15: X-181, 16: Negative control (H 2 O). B: Simultaneous RT-PCR amplification of the eight segments of A/California/07/2009 (H1N1) vaccine viruses using Thermo Scientist Extensor Mix, 1: 1 kb DNA Ladder, 2: X-181-M1, 3: X-179A-M1, 4: X-181-M2, 5: X-179A-M2, 6: X-181-M3, 7: X-179A-M4, 8: Negative control (H 2 O), 9: 1 kb DNA Ladder, 10: 121XP-M4, 11: X-181-M4, 12: X-179A-M3, 13: X-179A-M5, 14: X-179A, 15: X-181, 16: Negative control (H 2 O). C: The distribution amount of sequencing reads between the eight segments of influenza virus using different approaches of library preparation followed by Illumina HiSeq sequencing.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Negative Control, Sequencing

    4) Product Images from "Cellular reagents for diagnostics and synthetic biology"

    Article Title: Cellular reagents for diagnostics and synthetic biology

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201681

    PCR and Gibson assembly using cellular reagents. (a) Schematic depicting cellular PCR followed by cellular Gibson assembly for constructing new plasmids. Bacteria harboring target plasmids are mixed with polymerase-expressing cellular reagents and PCR is initiated by adding appropriate primers, buffer, and dNTP. The resulting PCR products are incubated with cellular reagents expressing Gibson assembly enzymes–Taq DNA polymerase, Taq DNA ligase, and T5 exonuclease–to assemble the new construct. (b) Cellular PCR amplification of vector and insert fragments directly from E . coli bacteria bearing target DNA plasmids using 2 x 10 7 cells of Phusion cellular reagents. Assembly parts include: (i) “pATetO 6XHis full length” vector for two part assembly with Kan r cassette bearing appropriate overlapping ends, and (ii) “pUC19 Fragments 1 and 2” for three part assembly with Kan r cassette whose ends overlap with pUC19 vector fragments. (c) Gibson assembly of agarose gel purified and unpurified cellular PCR products using pure or cellular Gibson assembly reagents. In “negative control” samples the PCR products were incubated in Gibson reaction buffer without pure or cellular Gibson enzymes. “pATetO 6XHis + Kan r ”represents a two part Gibson assembly while “Puc19 Fragment 1 + pUC19 Fragment 2 + Kan r ” represents a three-part Gibson assembly. Representative number of clones recovered in each case in the presence of both ampicillin and kanamycin are reported.
    Figure Legend Snippet: PCR and Gibson assembly using cellular reagents. (a) Schematic depicting cellular PCR followed by cellular Gibson assembly for constructing new plasmids. Bacteria harboring target plasmids are mixed with polymerase-expressing cellular reagents and PCR is initiated by adding appropriate primers, buffer, and dNTP. The resulting PCR products are incubated with cellular reagents expressing Gibson assembly enzymes–Taq DNA polymerase, Taq DNA ligase, and T5 exonuclease–to assemble the new construct. (b) Cellular PCR amplification of vector and insert fragments directly from E . coli bacteria bearing target DNA plasmids using 2 x 10 7 cells of Phusion cellular reagents. Assembly parts include: (i) “pATetO 6XHis full length” vector for two part assembly with Kan r cassette bearing appropriate overlapping ends, and (ii) “pUC19 Fragments 1 and 2” for three part assembly with Kan r cassette whose ends overlap with pUC19 vector fragments. (c) Gibson assembly of agarose gel purified and unpurified cellular PCR products using pure or cellular Gibson assembly reagents. In “negative control” samples the PCR products were incubated in Gibson reaction buffer without pure or cellular Gibson enzymes. “pATetO 6XHis + Kan r ”represents a two part Gibson assembly while “Puc19 Fragment 1 + pUC19 Fragment 2 + Kan r ” represents a three-part Gibson assembly. Representative number of clones recovered in each case in the presence of both ampicillin and kanamycin are reported.

    Techniques Used: Polymerase Chain Reaction, Expressing, Incubation, Construct, Amplification, Plasmid Preparation, Agarose Gel Electrophoresis, Purification, Clone Assay

    5) Product Images from "A Set of Assays for the Comprehensive Analysis of FMR1 Alleles in the Fragile X–Related Disorders"

    Article Title: A Set of Assays for the Comprehensive Analysis of FMR1 Alleles in the Fragile X–Related Disorders

    Journal: The Journal of Molecular Diagnostics : JMD

    doi: 10.1016/j.jmoldx.2016.06.001

    and either HiFi DNA polymerase or Phusion DNA polymerase. A: The products of the PCRs resolved by electrophoresis on a 1% agarose gel. Water (lane 1), DNA from a normal embryonic stem cell line (H1) (lane 2), from SC120, a PM induced pluripotent stem cell (iPSC) line (lane 3), from SC128, a FM iPSC line (lane 4), and from two FM cell lines, GM04025 (lane 5) and GM09237 (lane 6). The molecular weight marker (MW) was a 1-kb ladder. The asterisks indicate the repeat numbers corresponding to the amplified alleles. B: The electropheretograms produced on capillary electrophoresis for SC128 and GM09237 together with the LIZ1200 molecular weight marker. C: The results obtained for the amplification of DNA from a female (F) carrier of two normal alleles (30 repeats each), a male (M) carrier of a gray zone allele (GZ; 51 repeats), and their daughter who carries a normal allele and the paternal gray zone allele. Electrophoresis was performed on a 2% agarose gel.
    Figure Legend Snippet: and either HiFi DNA polymerase or Phusion DNA polymerase. A: The products of the PCRs resolved by electrophoresis on a 1% agarose gel. Water (lane 1), DNA from a normal embryonic stem cell line (H1) (lane 2), from SC120, a PM induced pluripotent stem cell (iPSC) line (lane 3), from SC128, a FM iPSC line (lane 4), and from two FM cell lines, GM04025 (lane 5) and GM09237 (lane 6). The molecular weight marker (MW) was a 1-kb ladder. The asterisks indicate the repeat numbers corresponding to the amplified alleles. B: The electropheretograms produced on capillary electrophoresis for SC128 and GM09237 together with the LIZ1200 molecular weight marker. C: The results obtained for the amplification of DNA from a female (F) carrier of two normal alleles (30 repeats each), a male (M) carrier of a gray zone allele (GZ; 51 repeats), and their daughter who carries a normal allele and the paternal gray zone allele. Electrophoresis was performed on a 2% agarose gel.

    Techniques Used: Electrophoresis, Agarose Gel Electrophoresis, Molecular Weight, Marker, Amplification, Produced

    6) Product Images from "Deep Sequencing for Evaluation of Genetic Stability of Influenza A/California/07/2009 (H1N1) Vaccine Viruses"

    Article Title: Deep Sequencing for Evaluation of Genetic Stability of Influenza A/California/07/2009 (H1N1) Vaccine Viruses

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0138650

    A: Simultaneous RT-PCR amplification of the eight segments of A/California/07/2009 (H1N1) vaccine viruses using Phusion DNA polymerase, 1: 1 kb DNA Ladder, 2: X-181-M1, 3: X-179A-M1, 4: X-181-M2, 5: X-179A-M2, 6: X-181-M3, 7: X-179A-M4, 8: 1 kb DNA Ladder, 9: 1 kb DNA Ladder, 10: 121XP-M4, 11: X-181-M4, 12: X-179A-M3, 13: X-179A-M5, 14: X-179A, 15: X-181, 16: Negative control (H 2 O). B: Simultaneous RT-PCR amplification of the eight segments of A/California/07/2009 (H1N1) vaccine viruses using Thermo Scientist Extensor Mix, 1: 1 kb DNA Ladder, 2: X-181-M1, 3: X-179A-M1, 4: X-181-M2, 5: X-179A-M2, 6: X-181-M3, 7: X-179A-M4, 8: Negative control (H 2 O), 9: 1 kb DNA Ladder, 10: 121XP-M4, 11: X-181-M4, 12: X-179A-M3, 13: X-179A-M5, 14: X-179A, 15: X-181, 16: Negative control (H 2 O). C: The distribution amount of sequencing reads between the eight segments of influenza virus using different approaches of library preparation followed by Illumina HiSeq sequencing.
    Figure Legend Snippet: A: Simultaneous RT-PCR amplification of the eight segments of A/California/07/2009 (H1N1) vaccine viruses using Phusion DNA polymerase, 1: 1 kb DNA Ladder, 2: X-181-M1, 3: X-179A-M1, 4: X-181-M2, 5: X-179A-M2, 6: X-181-M3, 7: X-179A-M4, 8: 1 kb DNA Ladder, 9: 1 kb DNA Ladder, 10: 121XP-M4, 11: X-181-M4, 12: X-179A-M3, 13: X-179A-M5, 14: X-179A, 15: X-181, 16: Negative control (H 2 O). B: Simultaneous RT-PCR amplification of the eight segments of A/California/07/2009 (H1N1) vaccine viruses using Thermo Scientist Extensor Mix, 1: 1 kb DNA Ladder, 2: X-181-M1, 3: X-179A-M1, 4: X-181-M2, 5: X-179A-M2, 6: X-181-M3, 7: X-179A-M4, 8: Negative control (H 2 O), 9: 1 kb DNA Ladder, 10: 121XP-M4, 11: X-181-M4, 12: X-179A-M3, 13: X-179A-M5, 14: X-179A, 15: X-181, 16: Negative control (H 2 O). C: The distribution amount of sequencing reads between the eight segments of influenza virus using different approaches of library preparation followed by Illumina HiSeq sequencing.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Negative Control, Sequencing

    7) Product Images from "Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates ▿"

    Article Title: Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates ▿

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.02340-10

    Genome replication, virion secretion, and protein expression of PCR clones derived from two thermophilic DNA polymerases. The 4B genome was amplified by 30 cycles of PCR using either High Fidelity plus DNA polymerase or Phusion DNA polymerase and then
    Figure Legend Snippet: Genome replication, virion secretion, and protein expression of PCR clones derived from two thermophilic DNA polymerases. The 4B genome was amplified by 30 cycles of PCR using either High Fidelity plus DNA polymerase or Phusion DNA polymerase and then

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

    8) Product Images from "Suggested Role for G4 DNA in Recombinational Switching at the Antigenic Variation Locus of the Lyme Disease Spirochete"

    Article Title: Suggested Role for G4 DNA in Recombinational Switching at the Antigenic Variation Locus of the Lyme Disease Spirochete

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0057792

    Precise excision of the vlsE variable region during PCR amplification. A) Schematic showing the variable, constant and 17 bp direct repeats of the vlsE gene. The location of PCR primers used for amplification are shown by arrows below the constant regions. B) An ethidium bromide-stained agarose gel showing amplification of a portion of vlsE with Phusion DNA polymerase using three different templates and two different primer sets. The templates used were B. burgdorferi B31 5A4 genomic DNA [29] pMBL20, a plasmid carrying the vlsE gene [51] ; the 776 bp or 935 bp PCR products resulting from PCR amplification. The asterisks indicate smaller discrete bands observed in lanes 1–3 and 4–6. M denotes a 100 bp molecular weight ladder marker. PCR samples were run on a 1.2% agarose gel in 1X TAE buffer at 80 V for 1.2 hours. C) Characterization of precise deletions in vlsE . The first entry on the left side of the panel shows the sequence obtained from direct sequencing of the PCR product obtained with either B248 and B249 or B1701 and 1702. The remainder of the lineup shows sequence generated with 10 randomly selected E. coli transformants. The transformants were generated by cloning the 223 bp truncated PCR product generated in vitro with primers B248 and B249. The fragment was gel-excised and cloned into pJET1.2/blunt vector (Fermentas). The alignment shows that all the sequenced vlsE inserts had a precise excision of the 570 bp variable region. D) Schematic showing the precise excision of the vlsE variable region that occurred during PCR amplification.
    Figure Legend Snippet: Precise excision of the vlsE variable region during PCR amplification. A) Schematic showing the variable, constant and 17 bp direct repeats of the vlsE gene. The location of PCR primers used for amplification are shown by arrows below the constant regions. B) An ethidium bromide-stained agarose gel showing amplification of a portion of vlsE with Phusion DNA polymerase using three different templates and two different primer sets. The templates used were B. burgdorferi B31 5A4 genomic DNA [29] pMBL20, a plasmid carrying the vlsE gene [51] ; the 776 bp or 935 bp PCR products resulting from PCR amplification. The asterisks indicate smaller discrete bands observed in lanes 1–3 and 4–6. M denotes a 100 bp molecular weight ladder marker. PCR samples were run on a 1.2% agarose gel in 1X TAE buffer at 80 V for 1.2 hours. C) Characterization of precise deletions in vlsE . The first entry on the left side of the panel shows the sequence obtained from direct sequencing of the PCR product obtained with either B248 and B249 or B1701 and 1702. The remainder of the lineup shows sequence generated with 10 randomly selected E. coli transformants. The transformants were generated by cloning the 223 bp truncated PCR product generated in vitro with primers B248 and B249. The fragment was gel-excised and cloned into pJET1.2/blunt vector (Fermentas). The alignment shows that all the sequenced vlsE inserts had a precise excision of the 570 bp variable region. D) Schematic showing the precise excision of the vlsE variable region that occurred during PCR amplification.

    Techniques Used: Polymerase Chain Reaction, Amplification, Staining, Agarose Gel Electrophoresis, Plasmid Preparation, Molecular Weight, Marker, Sequencing, Generated, Clone Assay, In Vitro

    Affect of mutations in the 17 bp direct repeats on precise excision of the vlsE variable region. A) DNA sequences of the wild-type 17 bp direct repeat (DR) and a mutant 17 bp direct repeat (DR*) used in this study. Mutated bases are highlighted in red. B) Schematic showing the plasmid templates carrying wild-type DRs and a mutant DR at the left, right or both sides of the variable region. C ) An ethidium bromide-stained agarose gel showing amplification of a portion of vlsE with Phusion DNA polymerase using the templates shown in Panel B with the indicated primers. Gel electrophoresis conditions were as noted in Fig. 2 .
    Figure Legend Snippet: Affect of mutations in the 17 bp direct repeats on precise excision of the vlsE variable region. A) DNA sequences of the wild-type 17 bp direct repeat (DR) and a mutant 17 bp direct repeat (DR*) used in this study. Mutated bases are highlighted in red. B) Schematic showing the plasmid templates carrying wild-type DRs and a mutant DR at the left, right or both sides of the variable region. C ) An ethidium bromide-stained agarose gel showing amplification of a portion of vlsE with Phusion DNA polymerase using the templates shown in Panel B with the indicated primers. Gel electrophoresis conditions were as noted in Fig. 2 .

    Techniques Used: Mutagenesis, Plasmid Preparation, Staining, Agarose Gel Electrophoresis, Amplification, Nucleic Acid Electrophoresis

    9) Product Images from "Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates ▿"

    Article Title: Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates ▿

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.02340-10

    Genome replication, virion secretion, and protein expression of PCR clones derived from two thermophilic DNA polymerases. The 4B genome was amplified by 30 cycles of PCR using either High Fidelity plus DNA polymerase or Phusion DNA polymerase and then
    Figure Legend Snippet: Genome replication, virion secretion, and protein expression of PCR clones derived from two thermophilic DNA polymerases. The 4B genome was amplified by 30 cycles of PCR using either High Fidelity plus DNA polymerase or Phusion DNA polymerase and then

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

    10) Product Images from "Restriction enzyme-free mutagenesis via the light regulation of DNA polymerization"

    Article Title: Restriction enzyme-free mutagenesis via the light regulation of DNA polymerization

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp150

    Effects of a caged thymidine nucleobase on the PCR. ( A ) PCR product using Phusion DNA Polymerase. Polymerization is halted in the presence of a caging group, which is confirmed using the truncated primer P3 that affords the same length product. ( B ) PCR product using Taq DNA polymerase. Polymerization is halted in the presence of a caging group, which is confirmed using the truncated primer P3 that affords the same length product.
    Figure Legend Snippet: Effects of a caged thymidine nucleobase on the PCR. ( A ) PCR product using Phusion DNA Polymerase. Polymerization is halted in the presence of a caging group, which is confirmed using the truncated primer P3 that affords the same length product. ( B ) PCR product using Taq DNA polymerase. Polymerization is halted in the presence of a caging group, which is confirmed using the truncated primer P3 that affords the same length product.

    Techniques Used: Polymerase Chain Reaction

    11) Product Images from "A Set of Assays for the Comprehensive Analysis of FMR1 Alleles in the Fragile X–Related Disorders"

    Article Title: A Set of Assays for the Comprehensive Analysis of FMR1 Alleles in the Fragile X–Related Disorders

    Journal: The Journal of Molecular Diagnostics : JMD

    doi: 10.1016/j.jmoldx.2016.06.001

    and either HiFi DNA polymerase or Phusion DNA polymerase. A: The products of the PCRs resolved by electrophoresis on a 1% agarose gel. Water (lane 1), DNA from a normal embryonic stem cell line (H1) (lane 2), from SC120, a PM induced pluripotent stem cell (iPSC) line (lane 3), from SC128, a FM iPSC line (lane 4), and from two FM cell lines, GM04025 (lane 5) and GM09237 (lane 6). The molecular weight marker (MW) was a 1-kb ladder. The asterisks indicate the repeat numbers corresponding to the amplified alleles. B: The electropheretograms produced on capillary electrophoresis for SC128 and GM09237 together with the LIZ1200 molecular weight marker. C: The results obtained for the amplification of DNA from a female (F) carrier of two normal alleles (30 repeats each), a male (M) carrier of a gray zone allele (GZ; 51 repeats), and their daughter who carries a normal allele and the paternal gray zone allele. Electrophoresis was performed on a 2% agarose gel.
    Figure Legend Snippet: and either HiFi DNA polymerase or Phusion DNA polymerase. A: The products of the PCRs resolved by electrophoresis on a 1% agarose gel. Water (lane 1), DNA from a normal embryonic stem cell line (H1) (lane 2), from SC120, a PM induced pluripotent stem cell (iPSC) line (lane 3), from SC128, a FM iPSC line (lane 4), and from two FM cell lines, GM04025 (lane 5) and GM09237 (lane 6). The molecular weight marker (MW) was a 1-kb ladder. The asterisks indicate the repeat numbers corresponding to the amplified alleles. B: The electropheretograms produced on capillary electrophoresis for SC128 and GM09237 together with the LIZ1200 molecular weight marker. C: The results obtained for the amplification of DNA from a female (F) carrier of two normal alleles (30 repeats each), a male (M) carrier of a gray zone allele (GZ; 51 repeats), and their daughter who carries a normal allele and the paternal gray zone allele. Electrophoresis was performed on a 2% agarose gel.

    Techniques Used: Electrophoresis, Agarose Gel Electrophoresis, Molecular Weight, Marker, Amplification, Produced

    12) Product Images from "Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates ▿"

    Article Title: Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates ▿

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.02340-10

    Genome replication, virion secretion, and protein expression of PCR clones derived from two thermophilic DNA polymerases. The 4B genome was amplified by 30 cycles of PCR using either High Fidelity plus DNA polymerase or Phusion DNA polymerase and then
    Figure Legend Snippet: Genome replication, virion secretion, and protein expression of PCR clones derived from two thermophilic DNA polymerases. The 4B genome was amplified by 30 cycles of PCR using either High Fidelity plus DNA polymerase or Phusion DNA polymerase and then

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

    13) Product Images from "Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates ▿"

    Article Title: Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates ▿

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.02340-10

    Genome replication, virion secretion, and protein expression of PCR clones derived from two thermophilic DNA polymerases. The 4B genome was amplified by 30 cycles of PCR using either High Fidelity plus DNA polymerase or Phusion DNA polymerase and then
    Figure Legend Snippet: Genome replication, virion secretion, and protein expression of PCR clones derived from two thermophilic DNA polymerases. The 4B genome was amplified by 30 cycles of PCR using either High Fidelity plus DNA polymerase or Phusion DNA polymerase and then

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

    14) Product Images from "A Set of Assays for the Comprehensive Analysis of FMR1 Alleles in the Fragile X–Related Disorders"

    Article Title: A Set of Assays for the Comprehensive Analysis of FMR1 Alleles in the Fragile X–Related Disorders

    Journal: The Journal of Molecular Diagnostics : JMD

    doi: 10.1016/j.jmoldx.2016.06.001

    and either HiFi DNA polymerase or Phusion DNA polymerase. A: The products of the PCRs resolved by electrophoresis on a 1% agarose gel. Water (lane 1), DNA from a normal embryonic stem cell line (H1) (lane 2), from SC120, a PM induced pluripotent stem cell (iPSC) line (lane 3), from SC128, a FM iPSC line (lane 4), and from two FM cell lines, GM04025 (lane 5) and GM09237 (lane 6). The molecular weight marker (MW) was a 1-kb ladder. The asterisks indicate the repeat numbers corresponding to the amplified alleles. B: The electropheretograms produced on capillary electrophoresis for SC128 and GM09237 together with the LIZ1200 molecular weight marker. C: The results obtained for the amplification of DNA from a female (F) carrier of two normal alleles (30 repeats each), a male (M) carrier of a gray zone allele (GZ; 51 repeats), and their daughter who carries a normal allele and the paternal gray zone allele. Electrophoresis was performed on a 2% agarose gel.
    Figure Legend Snippet: and either HiFi DNA polymerase or Phusion DNA polymerase. A: The products of the PCRs resolved by electrophoresis on a 1% agarose gel. Water (lane 1), DNA from a normal embryonic stem cell line (H1) (lane 2), from SC120, a PM induced pluripotent stem cell (iPSC) line (lane 3), from SC128, a FM iPSC line (lane 4), and from two FM cell lines, GM04025 (lane 5) and GM09237 (lane 6). The molecular weight marker (MW) was a 1-kb ladder. The asterisks indicate the repeat numbers corresponding to the amplified alleles. B: The electropheretograms produced on capillary electrophoresis for SC128 and GM09237 together with the LIZ1200 molecular weight marker. C: The results obtained for the amplification of DNA from a female (F) carrier of two normal alleles (30 repeats each), a male (M) carrier of a gray zone allele (GZ; 51 repeats), and their daughter who carries a normal allele and the paternal gray zone allele. Electrophoresis was performed on a 2% agarose gel.

    Techniques Used: Electrophoresis, Agarose Gel Electrophoresis, Molecular Weight, Marker, Amplification, Produced

    15) Product Images from "FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method"

    Article Title: FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-11-92

    Optimization of cloning conditions . (A) PCR amplification of a target cDNA (human nAChR α9 subunit) and the pGEMHE vector using different DNA polymerases: Pfu Turbo, PfuUltra and Phusion. (B) Comparison of number of colonies grown on the plates after transformation. Three different vector-to-insert ratios (1:1, 1:2, and 1:4) during Dpn I digestion and three amounts of vector-insert mixtures (2, 4, and 8 μl) for transformation were tested. See text for details. (C) Clone validation by PCR using GoTaq DNA polymerase. Lanes 1 to 12: target clones to be validated; Lane 13: 1 Kb plus DNA ladder; Lane 14: pGEMHE vector control; Lane 15: negative control using pCR4-TOPO-α9 parent plasmid. (D) Clone validation by restriction digestion to exclude unusual constructs. Lane 1: 1 Kb plus DNA ladder, Lanes 2-11: target clones double digested with Kpn I and Nhe I. Note that this digestion resulted in a pGEM vector and an insert with α9 nAChR plus the 5'UTR and 3'UTR of Xenopus β-globin.
    Figure Legend Snippet: Optimization of cloning conditions . (A) PCR amplification of a target cDNA (human nAChR α9 subunit) and the pGEMHE vector using different DNA polymerases: Pfu Turbo, PfuUltra and Phusion. (B) Comparison of number of colonies grown on the plates after transformation. Three different vector-to-insert ratios (1:1, 1:2, and 1:4) during Dpn I digestion and three amounts of vector-insert mixtures (2, 4, and 8 μl) for transformation were tested. See text for details. (C) Clone validation by PCR using GoTaq DNA polymerase. Lanes 1 to 12: target clones to be validated; Lane 13: 1 Kb plus DNA ladder; Lane 14: pGEMHE vector control; Lane 15: negative control using pCR4-TOPO-α9 parent plasmid. (D) Clone validation by restriction digestion to exclude unusual constructs. Lane 1: 1 Kb plus DNA ladder, Lanes 2-11: target clones double digested with Kpn I and Nhe I. Note that this digestion resulted in a pGEM vector and an insert with α9 nAChR plus the 5'UTR and 3'UTR of Xenopus β-globin.

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Transformation Assay, Negative Control, Construct

    16) Product Images from "Structural basis for targeted DNA cytosine deamination and mutagenesis by APOBEC3A and APOBEC3B"

    Article Title: Structural basis for targeted DNA cytosine deamination and mutagenesis by APOBEC3A and APOBEC3B

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.3344

    Deep-deamination approach to determine an optimal human A3A substrate A ssDNA library with a single target C and N’s on the 5′ and 3′ sides was reacted with human A3A (near-single hit kinetics). The resulting pool containing C-to-U deamination products was annealed to a bar-coded Illumina adaptor (IA) and T4 DNA polymerase was used to produce a complementary DNA strand. This intermediate was denatured, annealed to a 5′-IA, and converted to duplex by Phusion thermostable high fidelity DNA polymerase. Illumina Mi-Seq was used to generate reads for subsequent informatics analysis. A weblogo representation of deamination products unique to A3A shows enrichment for −1 T and +1 G that informed the ssDNA sequence for co-crystallization experiments (n=641; error bars are twice the sample correction value). Source data are provided as a spreadsheet (.xlsx) linked to the online legend.
    Figure Legend Snippet: Deep-deamination approach to determine an optimal human A3A substrate A ssDNA library with a single target C and N’s on the 5′ and 3′ sides was reacted with human A3A (near-single hit kinetics). The resulting pool containing C-to-U deamination products was annealed to a bar-coded Illumina adaptor (IA) and T4 DNA polymerase was used to produce a complementary DNA strand. This intermediate was denatured, annealed to a 5′-IA, and converted to duplex by Phusion thermostable high fidelity DNA polymerase. Illumina Mi-Seq was used to generate reads for subsequent informatics analysis. A weblogo representation of deamination products unique to A3A shows enrichment for −1 T and +1 G that informed the ssDNA sequence for co-crystallization experiments (n=641; error bars are twice the sample correction value). Source data are provided as a spreadsheet (.xlsx) linked to the online legend.

    Techniques Used: IA, Sequencing, Crystallization Assay

    17) Product Images from "Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates ▿"

    Article Title: Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates ▿

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.02340-10

    Genome replication, virion secretion, and protein expression of PCR clones derived from two thermophilic DNA polymerases. The 4B genome was amplified by 30 cycles of PCR using either High Fidelity plus DNA polymerase or Phusion DNA polymerase and then
    Figure Legend Snippet: Genome replication, virion secretion, and protein expression of PCR clones derived from two thermophilic DNA polymerases. The 4B genome was amplified by 30 cycles of PCR using either High Fidelity plus DNA polymerase or Phusion DNA polymerase and then

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

    18) Product Images from "Restriction enzyme-free mutagenesis via the light regulation of DNA polymerization"

    Article Title: Restriction enzyme-free mutagenesis via the light regulation of DNA polymerization

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp150

    Effects of a caged thymidine nucleobase on the PCR. ( A ) PCR product using Phusion DNA Polymerase. Polymerization is halted in the presence of a caging group, which is confirmed using the truncated primer P3 that affords the same length product. ( B ) PCR product using Taq DNA polymerase. Polymerization is halted in the presence of a caging group, which is confirmed using the truncated primer P3 that affords the same length product.
    Figure Legend Snippet: Effects of a caged thymidine nucleobase on the PCR. ( A ) PCR product using Phusion DNA Polymerase. Polymerization is halted in the presence of a caging group, which is confirmed using the truncated primer P3 that affords the same length product. ( B ) PCR product using Taq DNA polymerase. Polymerization is halted in the presence of a caging group, which is confirmed using the truncated primer P3 that affords the same length product.

    Techniques Used: Polymerase Chain Reaction

    19) Product Images from "Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates ▿"

    Article Title: Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates ▿

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.02340-10

    Genome replication, virion secretion, and protein expression of PCR clones derived from two thermophilic DNA polymerases. The 4B genome was amplified by 30 cycles of PCR using either High Fidelity plus DNA polymerase or Phusion DNA polymerase and then
    Figure Legend Snippet: Genome replication, virion secretion, and protein expression of PCR clones derived from two thermophilic DNA polymerases. The 4B genome was amplified by 30 cycles of PCR using either High Fidelity plus DNA polymerase or Phusion DNA polymerase and then

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

    20) Product Images from "Deep Sequencing for Evaluation of Genetic Stability of Influenza A/California/07/2009 (H1N1) Vaccine Viruses"

    Article Title: Deep Sequencing for Evaluation of Genetic Stability of Influenza A/California/07/2009 (H1N1) Vaccine Viruses

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0138650

    A: Simultaneous RT-PCR amplification of the eight segments of A/California/07/2009 (H1N1) vaccine viruses using Phusion DNA polymerase, 1: 1 kb DNA Ladder, 2: X-181-M1, 3: X-179A-M1, 4: X-181-M2, 5: X-179A-M2, 6: X-181-M3, 7: X-179A-M4, 8: 1 kb DNA Ladder, 9: 1 kb DNA Ladder, 10: 121XP-M4, 11: X-181-M4, 12: X-179A-M3, 13: X-179A-M5, 14: X-179A, 15: X-181, 16: Negative control (H 2 O). B: Simultaneous RT-PCR amplification of the eight segments of A/California/07/2009 (H1N1) vaccine viruses using Thermo Scientist Extensor Mix, 1: 1 kb DNA Ladder, 2: X-181-M1, 3: X-179A-M1, 4: X-181-M2, 5: X-179A-M2, 6: X-181-M3, 7: X-179A-M4, 8: Negative control (H 2 O), 9: 1 kb DNA Ladder, 10: 121XP-M4, 11: X-181-M4, 12: X-179A-M3, 13: X-179A-M5, 14: X-179A, 15: X-181, 16: Negative control (H 2 O). C: The distribution amount of sequencing reads between the eight segments of influenza virus using different approaches of library preparation followed by Illumina HiSeq sequencing.
    Figure Legend Snippet: A: Simultaneous RT-PCR amplification of the eight segments of A/California/07/2009 (H1N1) vaccine viruses using Phusion DNA polymerase, 1: 1 kb DNA Ladder, 2: X-181-M1, 3: X-179A-M1, 4: X-181-M2, 5: X-179A-M2, 6: X-181-M3, 7: X-179A-M4, 8: 1 kb DNA Ladder, 9: 1 kb DNA Ladder, 10: 121XP-M4, 11: X-181-M4, 12: X-179A-M3, 13: X-179A-M5, 14: X-179A, 15: X-181, 16: Negative control (H 2 O). B: Simultaneous RT-PCR amplification of the eight segments of A/California/07/2009 (H1N1) vaccine viruses using Thermo Scientist Extensor Mix, 1: 1 kb DNA Ladder, 2: X-181-M1, 3: X-179A-M1, 4: X-181-M2, 5: X-179A-M2, 6: X-181-M3, 7: X-179A-M4, 8: Negative control (H 2 O), 9: 1 kb DNA Ladder, 10: 121XP-M4, 11: X-181-M4, 12: X-179A-M3, 13: X-179A-M5, 14: X-179A, 15: X-181, 16: Negative control (H 2 O). C: The distribution amount of sequencing reads between the eight segments of influenza virus using different approaches of library preparation followed by Illumina HiSeq sequencing.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Negative Control, Sequencing

    21) Product Images from "Cellular reagents for diagnostics and synthetic biology"

    Article Title: Cellular reagents for diagnostics and synthetic biology

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201681

    PCR and Gibson assembly using cellular reagents. (a) Schematic depicting cellular PCR followed by cellular Gibson assembly for constructing new plasmids. Bacteria harboring target plasmids are mixed with polymerase-expressing cellular reagents and PCR is initiated by adding appropriate primers, buffer, and dNTP. The resulting PCR products are incubated with cellular reagents expressing Gibson assembly enzymes–Taq DNA polymerase, Taq DNA ligase, and T5 exonuclease–to assemble the new construct. (b) Cellular PCR amplification of vector and insert fragments directly from E . coli bacteria bearing target DNA plasmids using 2 x 10 7 cells of Phusion cellular reagents. Assembly parts include: (i) “pATetO 6XHis full length” vector for two part assembly with Kan r cassette bearing appropriate overlapping ends, and (ii) “pUC19 Fragments 1 and 2” for three part assembly with Kan r cassette whose ends overlap with pUC19 vector fragments. (c) Gibson assembly of agarose gel purified and unpurified cellular PCR products using pure or cellular Gibson assembly reagents. In “negative control” samples the PCR products were incubated in Gibson reaction buffer without pure or cellular Gibson enzymes. “pATetO 6XHis + Kan r ”represents a two part Gibson assembly while “Puc19 Fragment 1 + pUC19 Fragment 2 + Kan r ” represents a three-part Gibson assembly. Representative number of clones recovered in each case in the presence of both ampicillin and kanamycin are reported.
    Figure Legend Snippet: PCR and Gibson assembly using cellular reagents. (a) Schematic depicting cellular PCR followed by cellular Gibson assembly for constructing new plasmids. Bacteria harboring target plasmids are mixed with polymerase-expressing cellular reagents and PCR is initiated by adding appropriate primers, buffer, and dNTP. The resulting PCR products are incubated with cellular reagents expressing Gibson assembly enzymes–Taq DNA polymerase, Taq DNA ligase, and T5 exonuclease–to assemble the new construct. (b) Cellular PCR amplification of vector and insert fragments directly from E . coli bacteria bearing target DNA plasmids using 2 x 10 7 cells of Phusion cellular reagents. Assembly parts include: (i) “pATetO 6XHis full length” vector for two part assembly with Kan r cassette bearing appropriate overlapping ends, and (ii) “pUC19 Fragments 1 and 2” for three part assembly with Kan r cassette whose ends overlap with pUC19 vector fragments. (c) Gibson assembly of agarose gel purified and unpurified cellular PCR products using pure or cellular Gibson assembly reagents. In “negative control” samples the PCR products were incubated in Gibson reaction buffer without pure or cellular Gibson enzymes. “pATetO 6XHis + Kan r ”represents a two part Gibson assembly while “Puc19 Fragment 1 + pUC19 Fragment 2 + Kan r ” represents a three-part Gibson assembly. Representative number of clones recovered in each case in the presence of both ampicillin and kanamycin are reported.

    Techniques Used: Polymerase Chain Reaction, Expressing, Incubation, Construct, Amplification, Plasmid Preparation, Agarose Gel Electrophoresis, Purification, Clone Assay

    22) Product Images from "FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method"

    Article Title: FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-11-92

    Optimization of cloning conditions . (A) PCR amplification of a target cDNA (human nAChR α9 subunit) and the pGEMHE vector using different DNA polymerases: Pfu Turbo, PfuUltra and Phusion. (B) Comparison of number of colonies grown on the plates after transformation. Three different vector-to-insert ratios (1:1, 1:2, and 1:4) during Dpn I digestion and three amounts of vector-insert mixtures (2, 4, and 8 μl) for transformation were tested. See text for details. (C) Clone validation by PCR using GoTaq DNA polymerase. Lanes 1 to 12: target clones to be validated; Lane 13: 1 Kb plus DNA ladder; Lane 14: pGEMHE vector control; Lane 15: negative control using pCR4-TOPO-α9 parent plasmid. (D) Clone validation by restriction digestion to exclude unusual constructs. Lane 1: 1 Kb plus DNA ladder, Lanes 2-11: target clones double digested with Kpn I and Nhe I. Note that this digestion resulted in a pGEM vector and an insert with α9 nAChR plus the 5'UTR and 3'UTR of Xenopus β-globin.
    Figure Legend Snippet: Optimization of cloning conditions . (A) PCR amplification of a target cDNA (human nAChR α9 subunit) and the pGEMHE vector using different DNA polymerases: Pfu Turbo, PfuUltra and Phusion. (B) Comparison of number of colonies grown on the plates after transformation. Three different vector-to-insert ratios (1:1, 1:2, and 1:4) during Dpn I digestion and three amounts of vector-insert mixtures (2, 4, and 8 μl) for transformation were tested. See text for details. (C) Clone validation by PCR using GoTaq DNA polymerase. Lanes 1 to 12: target clones to be validated; Lane 13: 1 Kb plus DNA ladder; Lane 14: pGEMHE vector control; Lane 15: negative control using pCR4-TOPO-α9 parent plasmid. (D) Clone validation by restriction digestion to exclude unusual constructs. Lane 1: 1 Kb plus DNA ladder, Lanes 2-11: target clones double digested with Kpn I and Nhe I. Note that this digestion resulted in a pGEM vector and an insert with α9 nAChR plus the 5'UTR and 3'UTR of Xenopus β-globin.

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Transformation Assay, Negative Control, Construct

    23) Product Images from "A Set of Assays for the Comprehensive Analysis of FMR1 Alleles in the Fragile X–Related Disorders"

    Article Title: A Set of Assays for the Comprehensive Analysis of FMR1 Alleles in the Fragile X–Related Disorders

    Journal: The Journal of Molecular Diagnostics : JMD

    doi: 10.1016/j.jmoldx.2016.06.001

    and either HiFi DNA polymerase or Phusion DNA polymerase. A: The products of the PCRs resolved by electrophoresis on a 1% agarose gel. Water (lane 1), DNA from a normal embryonic stem cell line (H1) (lane 2), from SC120, a PM induced pluripotent stem cell (iPSC) line (lane 3), from SC128, a FM iPSC line (lane 4), and from two FM cell lines, GM04025 (lane 5) and GM09237 (lane 6). The molecular weight marker (MW) was a 1-kb ladder. The asterisks indicate the repeat numbers corresponding to the amplified alleles. B: The electropheretograms produced on capillary electrophoresis for SC128 and GM09237 together with the LIZ1200 molecular weight marker. C: The results obtained for the amplification of DNA from a female (F) carrier of two normal alleles (30 repeats each), a male (M) carrier of a gray zone allele (GZ; 51 repeats), and their daughter who carries a normal allele and the paternal gray zone allele. Electrophoresis was performed on a 2% agarose gel.
    Figure Legend Snippet: and either HiFi DNA polymerase or Phusion DNA polymerase. A: The products of the PCRs resolved by electrophoresis on a 1% agarose gel. Water (lane 1), DNA from a normal embryonic stem cell line (H1) (lane 2), from SC120, a PM induced pluripotent stem cell (iPSC) line (lane 3), from SC128, a FM iPSC line (lane 4), and from two FM cell lines, GM04025 (lane 5) and GM09237 (lane 6). The molecular weight marker (MW) was a 1-kb ladder. The asterisks indicate the repeat numbers corresponding to the amplified alleles. B: The electropheretograms produced on capillary electrophoresis for SC128 and GM09237 together with the LIZ1200 molecular weight marker. C: The results obtained for the amplification of DNA from a female (F) carrier of two normal alleles (30 repeats each), a male (M) carrier of a gray zone allele (GZ; 51 repeats), and their daughter who carries a normal allele and the paternal gray zone allele. Electrophoresis was performed on a 2% agarose gel.

    Techniques Used: Electrophoresis, Agarose Gel Electrophoresis, Molecular Weight, Marker, Amplification, Produced

    24) Product Images from "Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids"

    Article Title: Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids

    Journal: BioTechniques

    doi: 10.2144/000113418

    Analysis of the overlap extension PCR cloning reaction (A) Products of the overlap extension PCR cloning reaction after 0, 5, 10, 15, 20, 25, and 30 cycles by agarose gel electrophoresis. Three nanograms of pQE30 vector were mixed with 175 ng insert (250 molar excess) in 10 μL total volume; a 4-μL aliquot of reaction was separated on a 0.8% agarose gel. M2, 1 kb DNA ladder; M1, assembled plasmid in closed circular and relaxed circular forms. (B) Overlap extension PCR cloning efficiency of a gfp gene as a function of the number of PCR cycles. Twenty microliters of competent E. coli cells were transformed with 1 μL pQE30/insert overlap extension PCR. The number of green colonies was plotted against the number of PCR cycles for each plate. (C) Overlap extension PCR cloning efficiency as a function of the insert length. Phusion DNA polymerase was used to PCR-amplify products of various sizes: GFP ( gfp ) gene, β-D-glucuronidase ( gusA ) gene, β-galactosidase ( lacZ ) gene, and the luxABCDE operon from the carrying pIMBB plasmid. These products were gel-purified and used in the overlap extension PCR reaction with pQE30 vector. Three nanograms of pQE30 vector were mixed with 175–500 ng insert in a total reaction volume of 10 μL and subjected to 18 cycles of PCR. After Dpn I treatment, the overlap extension PCR products were used to transform competent E. coli cells. The number of colonies per plate was plotted against the size of the insert.
    Figure Legend Snippet: Analysis of the overlap extension PCR cloning reaction (A) Products of the overlap extension PCR cloning reaction after 0, 5, 10, 15, 20, 25, and 30 cycles by agarose gel electrophoresis. Three nanograms of pQE30 vector were mixed with 175 ng insert (250 molar excess) in 10 μL total volume; a 4-μL aliquot of reaction was separated on a 0.8% agarose gel. M2, 1 kb DNA ladder; M1, assembled plasmid in closed circular and relaxed circular forms. (B) Overlap extension PCR cloning efficiency of a gfp gene as a function of the number of PCR cycles. Twenty microliters of competent E. coli cells were transformed with 1 μL pQE30/insert overlap extension PCR. The number of green colonies was plotted against the number of PCR cycles for each plate. (C) Overlap extension PCR cloning efficiency as a function of the insert length. Phusion DNA polymerase was used to PCR-amplify products of various sizes: GFP ( gfp ) gene, β-D-glucuronidase ( gusA ) gene, β-galactosidase ( lacZ ) gene, and the luxABCDE operon from the carrying pIMBB plasmid. These products were gel-purified and used in the overlap extension PCR reaction with pQE30 vector. Three nanograms of pQE30 vector were mixed with 175–500 ng insert in a total reaction volume of 10 μL and subjected to 18 cycles of PCR. After Dpn I treatment, the overlap extension PCR products were used to transform competent E. coli cells. The number of colonies per plate was plotted against the size of the insert.

    Techniques Used: Polymerase Chain Reaction, Clone Assay, Agarose Gel Electrophoresis, Plasmid Preparation, Transformation Assay, Purification

    An outline of the overlap extension PCR cloning (A) First, the insert is PCR-amplified with the chimeric primers so that the final PCR product has overlapping regions with the vector. (B) Then, vector and insert are mixed, denatured and annealed; the hybridized insert then is extended by Phusion DNA polymerase using vector as a template until polymerase reaches 5′ end of the insert. After several PCR cycles, the new plasmid with two nicks (one on each strand) gets accumulated as a product. (C) The new plasmid can be transformed into E. coli after the parental plasmid is destroyed by Dpn I digest.
    Figure Legend Snippet: An outline of the overlap extension PCR cloning (A) First, the insert is PCR-amplified with the chimeric primers so that the final PCR product has overlapping regions with the vector. (B) Then, vector and insert are mixed, denatured and annealed; the hybridized insert then is extended by Phusion DNA polymerase using vector as a template until polymerase reaches 5′ end of the insert. After several PCR cycles, the new plasmid with two nicks (one on each strand) gets accumulated as a product. (C) The new plasmid can be transformed into E. coli after the parental plasmid is destroyed by Dpn I digest.

    Techniques Used: Polymerase Chain Reaction, Clone Assay, Amplification, Plasmid Preparation, Transformation Assay

    Related Articles

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    Article Title: Deep Sequencing for Evaluation of Genetic Stability of Influenza A/California/07/2009 (H1N1) Vaccine Viruses
    Article Snippet: .. This result demonstrated that adequate distribution of sequencing reads between segments were obtained from a DNA library prepared from samples amplified by Phusion DNA polymerase (DNA library), and by whole-RNA library. .. Consistency of Illumina sequencing, and sequencing analysis of A/PR/8/34 and A/California/07/2009 strains Center for Biologics Evaluation and Research (CBER) stock of A/PR/8/34 was kindly provided by Dr. Peter Palese at Mount.

    Article Title: Demonstration of the Presence of the “Deleted” MIR122 Gene in HepG2 Cells
    Article Snippet: .. Such changes were still observed after amplification with Phusion High Fidelity DNA Polymerase, with 3/15 (20%) of single allele clones obtained from HepG2 and Huh-7 DNA showing apparent poly(T) slippage and also four sequence variants observed that were not seen in other clones of the same haplotype ( ). .. Overall, despite this sequence heterogeneity, the polymorphisms confirmed two different haplotypes in HepG2 DNA, consistent with the presence of two alleles of the pre-mir-122 stem-loop region.

    Clone Assay:

    Article Title: Demonstration of the Presence of the “Deleted” MIR122 Gene in HepG2 Cells
    Article Snippet: .. Such changes were still observed after amplification with Phusion High Fidelity DNA Polymerase, with 3/15 (20%) of single allele clones obtained from HepG2 and Huh-7 DNA showing apparent poly(T) slippage and also four sequence variants observed that were not seen in other clones of the same haplotype ( ). .. Overall, despite this sequence heterogeneity, the polymorphisms confirmed two different haplotypes in HepG2 DNA, consistent with the presence of two alleles of the pre-mir-122 stem-loop region.

    Amplification:

    Article Title: Deep Sequencing for Evaluation of Genetic Stability of Influenza A/California/07/2009 (H1N1) Vaccine Viruses
    Article Snippet: .. This result demonstrated that adequate distribution of sequencing reads between segments were obtained from a DNA library prepared from samples amplified by Phusion DNA polymerase (DNA library), and by whole-RNA library. .. Consistency of Illumina sequencing, and sequencing analysis of A/PR/8/34 and A/California/07/2009 strains Center for Biologics Evaluation and Research (CBER) stock of A/PR/8/34 was kindly provided by Dr. Peter Palese at Mount.

    Article Title: Demonstration of the Presence of the “Deleted” MIR122 Gene in HepG2 Cells
    Article Snippet: .. Such changes were still observed after amplification with Phusion High Fidelity DNA Polymerase, with 3/15 (20%) of single allele clones obtained from HepG2 and Huh-7 DNA showing apparent poly(T) slippage and also four sequence variants observed that were not seen in other clones of the same haplotype ( ). .. Overall, despite this sequence heterogeneity, the polymorphisms confirmed two different haplotypes in HepG2 DNA, consistent with the presence of two alleles of the pre-mir-122 stem-loop region.

    Article Title: Fast and Reliable PCR Amplification from Aspergillus fumigatus Spore Suspension Without Traditional DNA Extraction). Fast and reliable PCR amplification from Aspergillus fumigatus spore suspension without traditional DNA extraction
    Article Snippet: .. Successful PCR amplification has also been obtained with a Phusion High‐Fidelity DNA polymerase (New England Biolabs; M0530; see Fig. A). .. This polymerase was tested in the following PCR conditions for PCR products of ∼1.2 kb: 1 cycle at 98°C for 30 s followed by 35 cycles of 98°C for 10 s, 58°C for 20 s, 72°C for 45 s, and finally 1 cycle of 72°C for 5 min. 8 Following the PCR, mix 5 µl of the PCR reaction with 1 µl of 6× DNA loading dye and load the reactions on an agarose gel (Voytas, ).

    DNA Purification:

    Article Title: Enzymatic Synthesis of Modified Oligonucleotides by PEAR Using Phusion and KOD DNA Polymerases
    Article Snippet: .. Four 2′-fluoro-2′-deoxyribinucleoside-5′-triphosphates (2′-F-dNTPs), including 2′-F-dATP, 2′-F-dCTP, 2′-F-dGTP, 2′-F-dUTP and four 2′-deoxyribonucleotides-5′-O-(1-thiotriphosphate) (dNTPαSs), including dATPαS, dGTPαS, dCTPαS, and dTTPαS, whose structural formula are shown in , were purchased from Trilink BioTechnologies, Inc. KOD DNA polymerase was purchased from TOYOBO (Shanghai) Biotech Co., Ltd. Phusion DNA polymerase, highly thermostable restriction enzyme PspGI, and dNTPs were purchased from New England Biolabs, Inc. UNIQ-10 Spin Column Oligo DNA Purification Kit was purchased from Sangon Biotech (Shanghai) Co., Ltd. .. Synthetic oligodeoxynucleotides, including a target ( X ) and a probe ( P ), were synthesized by Integrated DNA Technologies, Inc. and purified by high-performance liquid chromatography (HPLC).

    Polymerase Chain Reaction:

    Article Title: Variations of five eIF4E genes across cassava accessions exhibiting tolerant and susceptible responses to cassava brown streak disease
    Article Snippet: .. PCR was performed in a 20 μl reaction volume containing 10 unit Phusion DNA polymerase (NEB, Ipswich, MA), 1 μl of 1:5 diluted cDNA template, 1X Phusion PCR buffer, 5 μM each of upstream and downstream primers, and 250 nM dNTP with the following cycling condition: 98°C for 1 minute; 35 cycles of 98°C for 15 seconds, 56°C for 15 seconds, and 72°C for 45 seconds; and finally 72°C for 5 minutes. .. Primers were designed according to five annotated eIF4E transcripts identified in the draft cassava genomic sequence (Manihot esculenta v4.1) published in Phytozome ( http://phytozome.jgi.doe.gov ) in 2013, prior to the availability of the current cassava genome V6.1 ( ).

    Article Title: Fast and Reliable PCR Amplification from Aspergillus fumigatus Spore Suspension Without Traditional DNA Extraction). Fast and reliable PCR amplification from Aspergillus fumigatus spore suspension without traditional DNA extraction
    Article Snippet: .. Successful PCR amplification has also been obtained with a Phusion High‐Fidelity DNA polymerase (New England Biolabs; M0530; see Fig. A). .. This polymerase was tested in the following PCR conditions for PCR products of ∼1.2 kb: 1 cycle at 98°C for 30 s followed by 35 cycles of 98°C for 10 s, 58°C for 20 s, 72°C for 45 s, and finally 1 cycle of 72°C for 5 min. 8 Following the PCR, mix 5 µl of the PCR reaction with 1 µl of 6× DNA loading dye and load the reactions on an agarose gel (Voytas, ).

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    New England Biolabs phusion high fidelity dna polymerase
    The median CEL intensities for each amplicon obtained by using Stoffel <t>DNA</t> polymerase and <t>Phusion</t> DNA polymerase in the gap-fill reaction are plotted against each other. The CEL intensities that were
    Phusion High Fidelity Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 3841 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phusion high fidelity dna polymerase/product/New England Biolabs
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    New England Biolabs t4 ligase
    Schematic overview of the QL cloning procedure. An envelope gene or an envelope library is amplified with primers to introduce flanking Esp3I restriction sites enabling the generation of a 5′ NcoI and a 3′ Xho sitey (A; top). The envelope gene or an envelope library is incubated together with pQL9/11 in a one-tube reaction with Esp3I and <t>T4-Ligase.</t> Compatible “sticky-ends” (equally colored) can be ligated successfully, direct proper orientation and mediating resistance for further cleavage (A). Following transformation of CcdB sensitive bacteria, only recipients bearing a plasmid without CcdB are able to form colonies in the presence of ampicillin. (B) The lentiviral vector construct pQL9 comprises (i) 5′LTR (Long terminal repeat), (ii) EF1α (human promotor), (iii) GFP (marker gene), (iv) an IRES (internal ribosome entry site), (v) a CcdB positive selection marker [58] , and (vi) a 3′LTR sequence.
    T4 Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 810 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    The median CEL intensities for each amplicon obtained by using Stoffel DNA polymerase and Phusion DNA polymerase in the gap-fill reaction are plotted against each other. The CEL intensities that were

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: A comprehensive assay for targeted multiplex amplification of human DNA sequences

    doi: 10.1073/pnas.0803240105

    Figure Lengend Snippet: The median CEL intensities for each amplicon obtained by using Stoffel DNA polymerase and Phusion DNA polymerase in the gap-fill reaction are plotted against each other. The CEL intensities that were

    Article Snippet: The extension was performed by addition of 0.4 units of Phusion High-Fidelity DNA Polymerase (New England Biolabs), 3 μl 1.0 mM dNTP, 5 units Ampligase (Epicenter Biotechnologies) in a 15-μl volume at 60°C for 15 min followed by 72°C for 15 min.

    Techniques: Amplification

    Polyacrylamide gel electrophoresis (PAGE) electrophoresis of the polymerase–endonuclease amplification reaction (PEAR) products. Lowercase letters (agct) represents unmodified dNTPs; uppercase letters (AGCT) represent modified dNTPs (2′-F-dNTPs or dNTPαSs). (A) PEAR by Phusion DNA polymerase using unmodified dNTPs, 2′-F-modified dATP and dGTP, respectively. Lane 1: 10-bp DNA ladder; lane 2: normal dNTPs; lane 3: 2′-F-dATP modified PEAR products; lane 4: control without PspGI; lane 5: control without Phusion DNA polymerase; lane 6: control without dATP; lane 7: 10-bp DNA ladder; lane 8: 2′-F-dGTP modified PEAR products; lane 9: control without PspGI; lane 10 : control without Phusion DNA polymerase; lane 11: control without dGTP. (B) PEAR by Phusion DNA polymerase using 2′-F-dCTP and 2′-F-dUTP. Lane 1: 2′-F-dCTP modified PEAR products; lane 2: control without PspGI; lane 3: control without Phusion DNA polymerase; lane 4: control without dCTP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dUTP modified PEAR products; lane 7: control without PspGI; lane 8: control without Phusion DNA polymerase; lane 9: control without dUTP; lane 10: 10-bp DNA ladder. (C) 2′-F-dATP and 2′-F-dGTP modified PEAR products as “seeds” for PEAR. Lane 1: 10-bp DNA ladder; lane 2: control without PspGI; lane 3: using 2′-F-dATP modified PEAR products as “seeds” for PEAR; lane 4: control without PspGI; lane 5: using 2′-F-dGTP modified PEAR products as seeds for PEAR. (D) 2′-F-dATP and 2′-F-dGTP modified PEAR products using KOD DNA polymerase. Lane 1: 2′-F-dATP modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without 2′-F-dATP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dGTP modified PEAR products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase ; lane 9: control without 2′-F-dGTP; lane 10: 10-bp DNA ladder. (E) PEAR amplification of 2′-F-dCTP and 2′-F-dUTP modified products using KOD DNA polymerase. Lane 1: 2′-F-dCTP modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without dCTP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dUTP modified PEAR products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without dUTP; lane 10: 10-bp DNA ladder. (F) PEAR amplification of dTTPαS modified and 2′-F-dATP+dGTPαS double modified PEAR products using KOD DNA polymerase. Lane 1: dTTPαS modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without dTTPαS; lane 5: 20 bp DNA ladder; lane 6: 2′-F-dATP and dGTPαS double modified PEAR amplified products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without 2′-F-dATP and dGTPαS; lane 10: 20-bp DNA ladder. (G) PEAR amplification of 2′-F-dATP+dCTPαS double modified and 2′-F-dATP+dTTPαS double modified PEAR products using KOD DNA polymerase. Lane 1: 2′-F-dATP+dCTPαS double modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without 2′-F-dATP and dCTPαS; lane 5: 20-bp DNA ladder; lane 6: 2′-F-dATP+dTTPαS double modified PEAR amplified products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without 2′-F-dGTP and dTTPαS; lane 10: 20-bp DNA ladder.

    Journal: Nucleic Acid Therapeutics

    Article Title: Enzymatic Synthesis of Modified Oligonucleotides by PEAR Using Phusion and KOD DNA Polymerases

    doi: 10.1089/nat.2014.0513

    Figure Lengend Snippet: Polyacrylamide gel electrophoresis (PAGE) electrophoresis of the polymerase–endonuclease amplification reaction (PEAR) products. Lowercase letters (agct) represents unmodified dNTPs; uppercase letters (AGCT) represent modified dNTPs (2′-F-dNTPs or dNTPαSs). (A) PEAR by Phusion DNA polymerase using unmodified dNTPs, 2′-F-modified dATP and dGTP, respectively. Lane 1: 10-bp DNA ladder; lane 2: normal dNTPs; lane 3: 2′-F-dATP modified PEAR products; lane 4: control without PspGI; lane 5: control without Phusion DNA polymerase; lane 6: control without dATP; lane 7: 10-bp DNA ladder; lane 8: 2′-F-dGTP modified PEAR products; lane 9: control without PspGI; lane 10 : control without Phusion DNA polymerase; lane 11: control without dGTP. (B) PEAR by Phusion DNA polymerase using 2′-F-dCTP and 2′-F-dUTP. Lane 1: 2′-F-dCTP modified PEAR products; lane 2: control without PspGI; lane 3: control without Phusion DNA polymerase; lane 4: control without dCTP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dUTP modified PEAR products; lane 7: control without PspGI; lane 8: control without Phusion DNA polymerase; lane 9: control without dUTP; lane 10: 10-bp DNA ladder. (C) 2′-F-dATP and 2′-F-dGTP modified PEAR products as “seeds” for PEAR. Lane 1: 10-bp DNA ladder; lane 2: control without PspGI; lane 3: using 2′-F-dATP modified PEAR products as “seeds” for PEAR; lane 4: control without PspGI; lane 5: using 2′-F-dGTP modified PEAR products as seeds for PEAR. (D) 2′-F-dATP and 2′-F-dGTP modified PEAR products using KOD DNA polymerase. Lane 1: 2′-F-dATP modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without 2′-F-dATP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dGTP modified PEAR products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase ; lane 9: control without 2′-F-dGTP; lane 10: 10-bp DNA ladder. (E) PEAR amplification of 2′-F-dCTP and 2′-F-dUTP modified products using KOD DNA polymerase. Lane 1: 2′-F-dCTP modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without dCTP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dUTP modified PEAR products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without dUTP; lane 10: 10-bp DNA ladder. (F) PEAR amplification of dTTPαS modified and 2′-F-dATP+dGTPαS double modified PEAR products using KOD DNA polymerase. Lane 1: dTTPαS modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without dTTPαS; lane 5: 20 bp DNA ladder; lane 6: 2′-F-dATP and dGTPαS double modified PEAR amplified products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without 2′-F-dATP and dGTPαS; lane 10: 20-bp DNA ladder. (G) PEAR amplification of 2′-F-dATP+dCTPαS double modified and 2′-F-dATP+dTTPαS double modified PEAR products using KOD DNA polymerase. Lane 1: 2′-F-dATP+dCTPαS double modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without 2′-F-dATP and dCTPαS; lane 5: 20-bp DNA ladder; lane 6: 2′-F-dATP+dTTPαS double modified PEAR amplified products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without 2′-F-dGTP and dTTPαS; lane 10: 20-bp DNA ladder.

    Article Snippet: Four 2′-fluoro-2′-deoxyribinucleoside-5′-triphosphates (2′-F-dNTPs), including 2′-F-dATP, 2′-F-dCTP, 2′-F-dGTP, 2′-F-dUTP and four 2′-deoxyribonucleotides-5′-O-(1-thiotriphosphate) (dNTPαSs), including dATPαS, dGTPαS, dCTPαS, and dTTPαS, whose structural formula are shown in , were purchased from Trilink BioTechnologies, Inc. KOD DNA polymerase was purchased from TOYOBO (Shanghai) Biotech Co., Ltd. Phusion DNA polymerase, highly thermostable restriction enzyme PspGI, and dNTPs were purchased from New England Biolabs, Inc. UNIQ-10 Spin Column Oligo DNA Purification Kit was purchased from Sangon Biotech (Shanghai) Co., Ltd.

    Techniques: Polyacrylamide Gel Electrophoresis, Electrophoresis, Amplification, Modification

    RT-PCR amplification of five cassava eIF4E ORFs with gene-specific primers. Total RNA was extracted from TMS60444 cassava line. The first-strand cDNA was synthesized using SuperScript ™ III Reverse Transcriptase (Invitrogen) and PCR was performed using Phusion High-Fidelity DNA polymerase (New England Biolabs) and primers indicated in Table 1 . Lane 1: DNA marker; Lane 2: 016601 CDS amplified with primers 016601F and 016601R; Lane 3: 016620 CDS amplified with primers 016620F and 016620R; Lane 4: 015501 CDS amplified with 015501F and 015501R, Lane 5: 013223 CDS amplified with primers 013223lF and 013223R, Lane 6, 013223 CDS amplified with primers 013223sF and 013223R; Lane 7: 013732 CDS amplified with 013732lF and 013732R; Lane 8: 013732 CDS amplified with 013732sF and 013732R; and Lane 9: negative water control.

    Journal: PLoS ONE

    Article Title: Variations of five eIF4E genes across cassava accessions exhibiting tolerant and susceptible responses to cassava brown streak disease

    doi: 10.1371/journal.pone.0181998

    Figure Lengend Snippet: RT-PCR amplification of five cassava eIF4E ORFs with gene-specific primers. Total RNA was extracted from TMS60444 cassava line. The first-strand cDNA was synthesized using SuperScript ™ III Reverse Transcriptase (Invitrogen) and PCR was performed using Phusion High-Fidelity DNA polymerase (New England Biolabs) and primers indicated in Table 1 . Lane 1: DNA marker; Lane 2: 016601 CDS amplified with primers 016601F and 016601R; Lane 3: 016620 CDS amplified with primers 016620F and 016620R; Lane 4: 015501 CDS amplified with 015501F and 015501R, Lane 5: 013223 CDS amplified with primers 013223lF and 013223R, Lane 6, 013223 CDS amplified with primers 013223sF and 013223R; Lane 7: 013732 CDS amplified with 013732lF and 013732R; Lane 8: 013732 CDS amplified with 013732sF and 013732R; and Lane 9: negative water control.

    Article Snippet: PCR was performed in a 20 μl reaction volume containing 10 unit Phusion DNA polymerase (NEB, Ipswich, MA), 1 μl of 1:5 diluted cDNA template, 1X Phusion PCR buffer, 5 μM each of upstream and downstream primers, and 250 nM dNTP with the following cycling condition: 98°C for 1 minute; 35 cycles of 98°C for 15 seconds, 56°C for 15 seconds, and 72°C for 45 seconds; and finally 72°C for 5 minutes.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Synthesized, Polymerase Chain Reaction, Marker

    Schematic overview of the QL cloning procedure. An envelope gene or an envelope library is amplified with primers to introduce flanking Esp3I restriction sites enabling the generation of a 5′ NcoI and a 3′ Xho sitey (A; top). The envelope gene or an envelope library is incubated together with pQL9/11 in a one-tube reaction with Esp3I and T4-Ligase. Compatible “sticky-ends” (equally colored) can be ligated successfully, direct proper orientation and mediating resistance for further cleavage (A). Following transformation of CcdB sensitive bacteria, only recipients bearing a plasmid without CcdB are able to form colonies in the presence of ampicillin. (B) The lentiviral vector construct pQL9 comprises (i) 5′LTR (Long terminal repeat), (ii) EF1α (human promotor), (iii) GFP (marker gene), (iv) an IRES (internal ribosome entry site), (v) a CcdB positive selection marker [58] , and (vi) a 3′LTR sequence.

    Journal: PLoS ONE

    Article Title: A Mammalian Cell Based FACS-Panning Platform for the Selection of HIV-1 Envelopes for Vaccine Development

    doi: 10.1371/journal.pone.0109196

    Figure Lengend Snippet: Schematic overview of the QL cloning procedure. An envelope gene or an envelope library is amplified with primers to introduce flanking Esp3I restriction sites enabling the generation of a 5′ NcoI and a 3′ Xho sitey (A; top). The envelope gene or an envelope library is incubated together with pQL9/11 in a one-tube reaction with Esp3I and T4-Ligase. Compatible “sticky-ends” (equally colored) can be ligated successfully, direct proper orientation and mediating resistance for further cleavage (A). Following transformation of CcdB sensitive bacteria, only recipients bearing a plasmid without CcdB are able to form colonies in the presence of ampicillin. (B) The lentiviral vector construct pQL9 comprises (i) 5′LTR (Long terminal repeat), (ii) EF1α (human promotor), (iii) GFP (marker gene), (iv) an IRES (internal ribosome entry site), (v) a CcdB positive selection marker [58] , and (vi) a 3′LTR sequence.

    Article Snippet: Meanwhile a second reaction for the ligation was prepared. (II) 3 µL 10 mM ATP, 1 µL 10 x Tango Buffer, 1 µL 10 mM DTT, 1 µL T4-Ligase (NEB) addition of H2 0 to reach 10 µL.

    Techniques: Clone Assay, Amplification, Introduce, Incubation, Transformation Assay, Plasmid Preparation, Construct, Marker, Selection, Sequencing