q5 polymerase  (New England Biolabs)


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    Structured Review

    New England Biolabs q5 polymerase
    Q5 Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 170 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/q5 polymerase/product/New England Biolabs
    Average 99 stars, based on 170 article reviews
    Price from $9.99 to $1999.99
    q5 polymerase - by Bioz Stars, 2020-02
    99/100 stars

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    Related Articles

    Clone Assay:

    Article Title: RGS7 is recurrently mutated in melanoma and promotes migration and invasion of human cancer cells
    Article Snippet: PCR products were cloned into the pCDF1-MCS2-EF1-Puro vector via the BamHI and NotI restriction sites. .. The p.V56C, point mutation was introduced into the p.R44C by synthesizing the full vector using Q5 polymerase (NEB), phosphorylating the PCR product using PNK (NEB) and self ligating it using Quick Ligase (NEB).

    Article Title: The interaction of p130Cas with PKN3 promotes malignant growth
    Article Snippet: 2.3 Plasmid construction cDNA coding for p130Cas SRD domain variants (WT, 15AN; sequences provided in ) were commercially synthesized and cloned into the pMA‐T vector (geneArt, Thermo Fisher Scientific). .. Mutant variant S432A (KRLS to KRLA) was created subsequently by whole plasmid synthesis using Q5 polymerase (New England Biolabs, Ipswich, MA, USA) and respective site‐directed mutagenesis primers (listed in ).

    Amplification:

    Article Title: Structural characterization of CAS SH3 domain selectivity and regulation reveals new CAS interaction partners
    Article Snippet: In parallel, GLIS2 cDNA was prepared from the BLM melanoma cell line and amplified by PCR using the following primers: forward 5′-TA CTCGAG ATGGACTACAAAGACGATGACGACAAGCACTCCCTGGACGAGCCG-3′, reverse 5′-TA GAATTC TCAGTTCACCACAGCCGGT-3′. .. Site-directed mutagenesis of DOK7 and GLIS2 was performed by whole plasmid synthesis using Q5 polymerase (New England Biolabs) with respective primers (DOK7 mPR: forward 5′-TGGTGGGTGCCTCAAGGCCAGCACCGGTAGCGCTGCGTCCGCGG-3′, reverse 5′-CCGCGGACGCAGCGCTACCGGTGCTGGCCTTGAGGCACCCACCA-3′; GLIS2 mPR: forward 5′-CTCCTGCAGCTGCGCCCAGCACCGGTAGCGCCACTGCCCGCC-3′, reverse 5′-GGCGGGCAGTGGCGCTACCGGTGCTGGGCGCAGCTGCAGGAG-3′). pIRES2-FLAG-DOK7 and pIRES2-FLAG-GLIS2 were used as templates.

    Binding Assay:

    Article Title: Structural characterization of CAS SH3 domain selectivity and regulation reveals new CAS interaction partners
    Article Snippet: The chimeric CAS SH3 domains with the binding peptide were prepared by fusion of mouse CAS SH3 domain with a flexible spacer sequence (SGGSGSG) and binding peptides derived from PTP-PEST (327–343 of mouse PTP-PEST, UniProtKB accession number P35831) or Vinculin (854–870 of mouse Vinculin, UniProtKB accession number Q64727). cDNA coding for the flexible peptides with the binding peptides was commercially synthesized (geneArt, Life technologies) and inserted in frame at the 3′ end of the CAS SH3 domain using Bam HI/Eco RI sites. .. Site-directed mutagenesis of DOK7 and GLIS2 was performed by whole plasmid synthesis using Q5 polymerase (New England Biolabs) with respective primers (DOK7 mPR: forward 5′-TGGTGGGTGCCTCAAGGCCAGCACCGGTAGCGCTGCGTCCGCGG-3′, reverse 5′-CCGCGGACGCAGCGCTACCGGTGCTGGCCTTGAGGCACCCACCA-3′; GLIS2 mPR: forward 5′-CTCCTGCAGCTGCGCCCAGCACCGGTAGCGCCACTGCCCGCC-3′, reverse 5′-GGCGGGCAGTGGCGCTACCGGTGCTGGGCGCAGCTGCAGGAG-3′). pIRES2-FLAG-DOK7 and pIRES2-FLAG-GLIS2 were used as templates.

    Synthesized:

    Article Title: Structural characterization of CAS SH3 domain selectivity and regulation reveals new CAS interaction partners
    Article Snippet: The chimeric CAS SH3 domains with the binding peptide were prepared by fusion of mouse CAS SH3 domain with a flexible spacer sequence (SGGSGSG) and binding peptides derived from PTP-PEST (327–343 of mouse PTP-PEST, UniProtKB accession number P35831) or Vinculin (854–870 of mouse Vinculin, UniProtKB accession number Q64727). cDNA coding for the flexible peptides with the binding peptides was commercially synthesized (geneArt, Life technologies) and inserted in frame at the 3′ end of the CAS SH3 domain using Bam HI/Eco RI sites. .. Site-directed mutagenesis of DOK7 and GLIS2 was performed by whole plasmid synthesis using Q5 polymerase (New England Biolabs) with respective primers (DOK7 mPR: forward 5′-TGGTGGGTGCCTCAAGGCCAGCACCGGTAGCGCTGCGTCCGCGG-3′, reverse 5′-CCGCGGACGCAGCGCTACCGGTGCTGGCCTTGAGGCACCCACCA-3′; GLIS2 mPR: forward 5′-CTCCTGCAGCTGCGCCCAGCACCGGTAGCGCCACTGCCCGCC-3′, reverse 5′-GGCGGGCAGTGGCGCTACCGGTGCTGGGCGCAGCTGCAGGAG-3′). pIRES2-FLAG-DOK7 and pIRES2-FLAG-GLIS2 were used as templates.

    Article Title: The interaction of p130Cas with PKN3 promotes malignant growth
    Article Snippet: 2.3 Plasmid construction cDNA coding for p130Cas SRD domain variants (WT, 15AN; sequences provided in ) were commercially synthesized and cloned into the pMA‐T vector (geneArt, Thermo Fisher Scientific). .. Mutant variant S432A (KRLS to KRLA) was created subsequently by whole plasmid synthesis using Q5 polymerase (New England Biolabs, Ipswich, MA, USA) and respective site‐directed mutagenesis primers (listed in ).

    Mutagenesis:

    Article Title: Structural characterization of CAS SH3 domain selectivity and regulation reveals new CAS interaction partners
    Article Snippet: .. Site-directed mutagenesis of DOK7 and GLIS2 was performed by whole plasmid synthesis using Q5 polymerase (New England Biolabs) with respective primers (DOK7 mPR: forward 5′-TGGTGGGTGCCTCAAGGCCAGCACCGGTAGCGCTGCGTCCGCGG-3′, reverse 5′-CCGCGGACGCAGCGCTACCGGTGCTGGCCTTGAGGCACCCACCA-3′; GLIS2 mPR: forward 5′-CTCCTGCAGCTGCGCCCAGCACCGGTAGCGCCACTGCCCGCC-3′, reverse 5′-GGCGGGCAGTGGCGCTACCGGTGCTGGGCGCAGCTGCAGGAG-3′). pIRES2-FLAG-DOK7 and pIRES2-FLAG-GLIS2 were used as templates. .. After PCR, 5U of Dpn I were added to each reaction and incubated for 1.5 h at 37 °C.

    Article Title: RGS7 is recurrently mutated in melanoma and promotes migration and invasion of human cancer cells
    Article Snippet: .. The p.V56C, point mutation was introduced into the p.R44C by synthesizing the full vector using Q5 polymerase (NEB), phosphorylating the PCR product using PNK (NEB) and self ligating it using Quick Ligase (NEB). ..

    Article Title: The interaction of p130Cas with PKN3 promotes malignant growth
    Article Snippet: .. Mutant variant S432A (KRLS to KRLA) was created subsequently by whole plasmid synthesis using Q5 polymerase (New England Biolabs, Ipswich, MA, USA) and respective site‐directed mutagenesis primers (listed in ). .. Following PCR, 5U of DpnI was added to each reaction and incubated for 1.5 h at 37 °C.

    Isolation:

    Article Title: Structural characterization of CAS SH3 domain selectivity and regulation reveals new CAS interaction partners
    Article Snippet: DOK7 cDNA was PCR-amplified from cDNA isolated from MCF7 human breast carcinoma cells using a forward primer (5′-TA CTCGAG ATGGACTACAAAGACGATGACGACAAGAAGATGACCGAGGCGGCG-3′) that includes a sequence coding for Flag epitope and a reverse primer (5′-TA GAATTC GCTCTCAAGGAGGGGGGTTTACC-3′). .. Site-directed mutagenesis of DOK7 and GLIS2 was performed by whole plasmid synthesis using Q5 polymerase (New England Biolabs) with respective primers (DOK7 mPR: forward 5′-TGGTGGGTGCCTCAAGGCCAGCACCGGTAGCGCTGCGTCCGCGG-3′, reverse 5′-CCGCGGACGCAGCGCTACCGGTGCTGGCCTTGAGGCACCCACCA-3′; GLIS2 mPR: forward 5′-CTCCTGCAGCTGCGCCCAGCACCGGTAGCGCCACTGCCCGCC-3′, reverse 5′-GGCGGGCAGTGGCGCTACCGGTGCTGGGCGCAGCTGCAGGAG-3′). pIRES2-FLAG-DOK7 and pIRES2-FLAG-GLIS2 were used as templates.

    Incubation:

    Article Title: Structural characterization of CAS SH3 domain selectivity and regulation reveals new CAS interaction partners
    Article Snippet: Site-directed mutagenesis of DOK7 and GLIS2 was performed by whole plasmid synthesis using Q5 polymerase (New England Biolabs) with respective primers (DOK7 mPR: forward 5′-TGGTGGGTGCCTCAAGGCCAGCACCGGTAGCGCTGCGTCCGCGG-3′, reverse 5′-CCGCGGACGCAGCGCTACCGGTGCTGGCCTTGAGGCACCCACCA-3′; GLIS2 mPR: forward 5′-CTCCTGCAGCTGCGCCCAGCACCGGTAGCGCCACTGCCCGCC-3′, reverse 5′-GGCGGGCAGTGGCGCTACCGGTGCTGGGCGCAGCTGCAGGAG-3′). pIRES2-FLAG-DOK7 and pIRES2-FLAG-GLIS2 were used as templates. .. After PCR, 5U of Dpn I were added to each reaction and incubated for 1.5 h at 37 °C.

    Article Title: The interaction of p130Cas with PKN3 promotes malignant growth
    Article Snippet: Mutant variant S432A (KRLS to KRLA) was created subsequently by whole plasmid synthesis using Q5 polymerase (New England Biolabs, Ipswich, MA, USA) and respective site‐directed mutagenesis primers (listed in ). .. Following PCR, 5U of DpnI was added to each reaction and incubated for 1.5 h at 37 °C.

    Construct:

    Article Title: Structural characterization of CAS SH3 domain selectivity and regulation reveals new CAS interaction partners
    Article Snippet: Plasmid construction pEGFP-C1 CAS and pGEX-CAS-SH3 domain constructs were prepared previously . .. Site-directed mutagenesis of DOK7 and GLIS2 was performed by whole plasmid synthesis using Q5 polymerase (New England Biolabs) with respective primers (DOK7 mPR: forward 5′-TGGTGGGTGCCTCAAGGCCAGCACCGGTAGCGCTGCGTCCGCGG-3′, reverse 5′-CCGCGGACGCAGCGCTACCGGTGCTGGCCTTGAGGCACCCACCA-3′; GLIS2 mPR: forward 5′-CTCCTGCAGCTGCGCCCAGCACCGGTAGCGCCACTGCCCGCC-3′, reverse 5′-GGCGGGCAGTGGCGCTACCGGTGCTGGGCGCAGCTGCAGGAG-3′). pIRES2-FLAG-DOK7 and pIRES2-FLAG-GLIS2 were used as templates.

    Article Title: The interaction of p130Cas with PKN3 promotes malignant growth
    Article Snippet: Mutant variant S432A (KRLS to KRLA) was created subsequently by whole plasmid synthesis using Q5 polymerase (New England Biolabs, Ipswich, MA, USA) and respective site‐directed mutagenesis primers (listed in ). .. Sequences of all SRD variants were then switched with the original p130Cas SRD domain cassette within the p130Cas sequence (pUC19 vector) using Bpu10I/SacII sites. pEGFP‐C1 p130Cas variants and pGEX‐p130Cas‐SH3 domain constructs were prepared similarly as described previously (Braniš et al ., ; Janoštiak et al ., ).

    FLAG-tag:

    Article Title: Structural characterization of CAS SH3 domain selectivity and regulation reveals new CAS interaction partners
    Article Snippet: DOK7 cDNA was PCR-amplified from cDNA isolated from MCF7 human breast carcinoma cells using a forward primer (5′-TA CTCGAG ATGGACTACAAAGACGATGACGACAAGAAGATGACCGAGGCGGCG-3′) that includes a sequence coding for Flag epitope and a reverse primer (5′-TA GAATTC GCTCTCAAGGAGGGGGGTTTACC-3′). .. Site-directed mutagenesis of DOK7 and GLIS2 was performed by whole plasmid synthesis using Q5 polymerase (New England Biolabs) with respective primers (DOK7 mPR: forward 5′-TGGTGGGTGCCTCAAGGCCAGCACCGGTAGCGCTGCGTCCGCGG-3′, reverse 5′-CCGCGGACGCAGCGCTACCGGTGCTGGCCTTGAGGCACCCACCA-3′; GLIS2 mPR: forward 5′-CTCCTGCAGCTGCGCCCAGCACCGGTAGCGCCACTGCCCGCC-3′, reverse 5′-GGCGGGCAGTGGCGCTACCGGTGCTGGGCGCAGCTGCAGGAG-3′). pIRES2-FLAG-DOK7 and pIRES2-FLAG-GLIS2 were used as templates.

    Article Title: RGS7 is recurrently mutated in melanoma and promotes migration and invasion of human cancer cells
    Article Snippet: A FLAG tag was introduced onto the C-terminus of RGS7 during the cloning procedure. .. The p.V56C, point mutation was introduced into the p.R44C by synthesizing the full vector using Q5 polymerase (NEB), phosphorylating the PCR product using PNK (NEB) and self ligating it using Quick Ligase (NEB).

    Generated:

    Article Title: The interaction of p130Cas with PKN3 promotes malignant growth
    Article Snippet: The 15AN variant was generated by mutation of all 15 Ser/Thr to Ala or Asn. .. Mutant variant S432A (KRLS to KRLA) was created subsequently by whole plasmid synthesis using Q5 polymerase (New England Biolabs, Ipswich, MA, USA) and respective site‐directed mutagenesis primers (listed in ).

    Polymerase Chain Reaction:

    Article Title: Structural characterization of CAS SH3 domain selectivity and regulation reveals new CAS interaction partners
    Article Snippet: The PCR-amplified DOK7 and GLIS2 cDNA were cleaved with Xho I/Eco RI and inserted into Xho I/Eco RI-cleaved pIRES2-EGFP (Addgene), resulting in plasmids pIRES2-FLAG-DOK7 and pIRES2-FLAG-GLIS2. .. Site-directed mutagenesis of DOK7 and GLIS2 was performed by whole plasmid synthesis using Q5 polymerase (New England Biolabs) with respective primers (DOK7 mPR: forward 5′-TGGTGGGTGCCTCAAGGCCAGCACCGGTAGCGCTGCGTCCGCGG-3′, reverse 5′-CCGCGGACGCAGCGCTACCGGTGCTGGCCTTGAGGCACCCACCA-3′; GLIS2 mPR: forward 5′-CTCCTGCAGCTGCGCCCAGCACCGGTAGCGCCACTGCCCGCC-3′, reverse 5′-GGCGGGCAGTGGCGCTACCGGTGCTGGGCGCAGCTGCAGGAG-3′). pIRES2-FLAG-DOK7 and pIRES2-FLAG-GLIS2 were used as templates.

    Article Title: RGS7 is recurrently mutated in melanoma and promotes migration and invasion of human cancer cells
    Article Snippet: .. The p.V56C, point mutation was introduced into the p.R44C by synthesizing the full vector using Q5 polymerase (NEB), phosphorylating the PCR product using PNK (NEB) and self ligating it using Quick Ligase (NEB). ..

    Article Title: The interaction of p130Cas with PKN3 promotes malignant growth
    Article Snippet: Mutant variant S432A (KRLS to KRLA) was created subsequently by whole plasmid synthesis using Q5 polymerase (New England Biolabs, Ipswich, MA, USA) and respective site‐directed mutagenesis primers (listed in ). .. Following PCR, 5U of DpnI was added to each reaction and incubated for 1.5 h at 37 °C.

    Plasmid Preparation:

    Article Title: Structural characterization of CAS SH3 domain selectivity and regulation reveals new CAS interaction partners
    Article Snippet: .. Site-directed mutagenesis of DOK7 and GLIS2 was performed by whole plasmid synthesis using Q5 polymerase (New England Biolabs) with respective primers (DOK7 mPR: forward 5′-TGGTGGGTGCCTCAAGGCCAGCACCGGTAGCGCTGCGTCCGCGG-3′, reverse 5′-CCGCGGACGCAGCGCTACCGGTGCTGGCCTTGAGGCACCCACCA-3′; GLIS2 mPR: forward 5′-CTCCTGCAGCTGCGCCCAGCACCGGTAGCGCCACTGCCCGCC-3′, reverse 5′-GGCGGGCAGTGGCGCTACCGGTGCTGGGCGCAGCTGCAGGAG-3′). pIRES2-FLAG-DOK7 and pIRES2-FLAG-GLIS2 were used as templates. .. After PCR, 5U of Dpn I were added to each reaction and incubated for 1.5 h at 37 °C.

    Article Title: RGS7 is recurrently mutated in melanoma and promotes migration and invasion of human cancer cells
    Article Snippet: .. The p.V56C, point mutation was introduced into the p.R44C by synthesizing the full vector using Q5 polymerase (NEB), phosphorylating the PCR product using PNK (NEB) and self ligating it using Quick Ligase (NEB). ..

    Article Title: The interaction of p130Cas with PKN3 promotes malignant growth
    Article Snippet: .. Mutant variant S432A (KRLS to KRLA) was created subsequently by whole plasmid synthesis using Q5 polymerase (New England Biolabs, Ipswich, MA, USA) and respective site‐directed mutagenesis primers (listed in ). .. Following PCR, 5U of DpnI was added to each reaction and incubated for 1.5 h at 37 °C.

    Expressing:

    Article Title: RGS7 is recurrently mutated in melanoma and promotes migration and invasion of human cancer cells
    Article Snippet: Paragraph title: Construction of wild-type and mutant expression vectors ... The p.V56C, point mutation was introduced into the p.R44C by synthesizing the full vector using Q5 polymerase (NEB), phosphorylating the PCR product using PNK (NEB) and self ligating it using Quick Ligase (NEB).

    Sequencing:

    Article Title: Structural characterization of CAS SH3 domain selectivity and regulation reveals new CAS interaction partners
    Article Snippet: DOK7 cDNA was PCR-amplified from cDNA isolated from MCF7 human breast carcinoma cells using a forward primer (5′-TA CTCGAG ATGGACTACAAAGACGATGACGACAAGAAGATGACCGAGGCGGCG-3′) that includes a sequence coding for Flag epitope and a reverse primer (5′-TA GAATTC GCTCTCAAGGAGGGGGGTTTACC-3′). .. Site-directed mutagenesis of DOK7 and GLIS2 was performed by whole plasmid synthesis using Q5 polymerase (New England Biolabs) with respective primers (DOK7 mPR: forward 5′-TGGTGGGTGCCTCAAGGCCAGCACCGGTAGCGCTGCGTCCGCGG-3′, reverse 5′-CCGCGGACGCAGCGCTACCGGTGCTGGCCTTGAGGCACCCACCA-3′; GLIS2 mPR: forward 5′-CTCCTGCAGCTGCGCCCAGCACCGGTAGCGCCACTGCCCGCC-3′, reverse 5′-GGCGGGCAGTGGCGCTACCGGTGCTGGGCGCAGCTGCAGGAG-3′). pIRES2-FLAG-DOK7 and pIRES2-FLAG-GLIS2 were used as templates.

    Variant Assay:

    Article Title: The interaction of p130Cas with PKN3 promotes malignant growth
    Article Snippet: .. Mutant variant S432A (KRLS to KRLA) was created subsequently by whole plasmid synthesis using Q5 polymerase (New England Biolabs, Ipswich, MA, USA) and respective site‐directed mutagenesis primers (listed in ). .. Following PCR, 5U of DpnI was added to each reaction and incubated for 1.5 h at 37 °C.

    Derivative Assay:

    Article Title: Structural characterization of CAS SH3 domain selectivity and regulation reveals new CAS interaction partners
    Article Snippet: The chimeric CAS SH3 domains with the binding peptide were prepared by fusion of mouse CAS SH3 domain with a flexible spacer sequence (SGGSGSG) and binding peptides derived from PTP-PEST (327–343 of mouse PTP-PEST, UniProtKB accession number P35831) or Vinculin (854–870 of mouse Vinculin, UniProtKB accession number Q64727). cDNA coding for the flexible peptides with the binding peptides was commercially synthesized (geneArt, Life technologies) and inserted in frame at the 3′ end of the CAS SH3 domain using Bam HI/Eco RI sites. .. Site-directed mutagenesis of DOK7 and GLIS2 was performed by whole plasmid synthesis using Q5 polymerase (New England Biolabs) with respective primers (DOK7 mPR: forward 5′-TGGTGGGTGCCTCAAGGCCAGCACCGGTAGCGCTGCGTCCGCGG-3′, reverse 5′-CCGCGGACGCAGCGCTACCGGTGCTGGCCTTGAGGCACCCACCA-3′; GLIS2 mPR: forward 5′-CTCCTGCAGCTGCGCCCAGCACCGGTAGCGCCACTGCCCGCC-3′, reverse 5′-GGCGGGCAGTGGCGCTACCGGTGCTGGGCGCAGCTGCAGGAG-3′). pIRES2-FLAG-DOK7 and pIRES2-FLAG-GLIS2 were used as templates.

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    New England Biolabs q5 dna polymerase
    PCR based verification of genome edit A. A schematic representation showing the locations of PCR primers used in panel B; B. Agarose gel stained with ethidium bromide of amplified PCR products using primers specific to the point mutation (H743A, for which we changed CAC→GCT) and the B. subtilis chromosome (left panel), and using primers specific to the editing plasmid (right panel). Of twelve isolates screened all were found to have the predicted point mutation, and two of twelve retained resistance to spectinomycin (asterisks). The slow migrating band in the PY79 lane corresponds to nonspecific PCR amplification in the wild type background. The editing plasmid was detectable in spectinomycin resistant isolates, but not in the remaining ten. C. Agarose gel stained with ethidium bromide of PCR reactions using primers specific for deletion of four B. subtilis genes. Primers were designed outside of the editing template, and are therefore specific to chromosomal DNA. For all four deletions, all isolates were found to be spectinomycin sensitive, and PCR genotyping of three isolates for each deletion found that all isolates tested had the intended deletion. Note that for the control reaction for ponA , the PCR product is faint because the amplicon is approximately 5 kb and the extension time of the PCR was 3 min, which is near the limit for complete extension for <t>Q5</t> DNA polymerase; the other three control amplicons are no larger than 3.4 kb.
    Q5 Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 718 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/q5 dna polymerase/product/New England Biolabs
    Average 95 stars, based on 718 article reviews
    Price from $9.99 to $1999.99
    q5 dna polymerase - by Bioz Stars, 2020-02
    95/100 stars
      Buy from Supplier

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    Q5U Hot Start High Fidelity DNA Polymerase is a modified version of Q5 High Fidelity DNA Polymerase which efficiently incorporates dUTP and amplifies uracil containing DNA templates This feature is
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    PCR based verification of genome edit A. A schematic representation showing the locations of PCR primers used in panel B; B. Agarose gel stained with ethidium bromide of amplified PCR products using primers specific to the point mutation (H743A, for which we changed CAC→GCT) and the B. subtilis chromosome (left panel), and using primers specific to the editing plasmid (right panel). Of twelve isolates screened all were found to have the predicted point mutation, and two of twelve retained resistance to spectinomycin (asterisks). The slow migrating band in the PY79 lane corresponds to nonspecific PCR amplification in the wild type background. The editing plasmid was detectable in spectinomycin resistant isolates, but not in the remaining ten. C. Agarose gel stained with ethidium bromide of PCR reactions using primers specific for deletion of four B. subtilis genes. Primers were designed outside of the editing template, and are therefore specific to chromosomal DNA. For all four deletions, all isolates were found to be spectinomycin sensitive, and PCR genotyping of three isolates for each deletion found that all isolates tested had the intended deletion. Note that for the control reaction for ponA , the PCR product is faint because the amplicon is approximately 5 kb and the extension time of the PCR was 3 min, which is near the limit for complete extension for Q5 DNA polymerase; the other three control amplicons are no larger than 3.4 kb.

    Journal: Bio-protocol

    Article Title: CRISPR/Cas9 Editing of the Bacillus subtilis Genome

    doi: 10.21769/BioProtoc.2272

    Figure Lengend Snippet: PCR based verification of genome edit A. A schematic representation showing the locations of PCR primers used in panel B; B. Agarose gel stained with ethidium bromide of amplified PCR products using primers specific to the point mutation (H743A, for which we changed CAC→GCT) and the B. subtilis chromosome (left panel), and using primers specific to the editing plasmid (right panel). Of twelve isolates screened all were found to have the predicted point mutation, and two of twelve retained resistance to spectinomycin (asterisks). The slow migrating band in the PY79 lane corresponds to nonspecific PCR amplification in the wild type background. The editing plasmid was detectable in spectinomycin resistant isolates, but not in the remaining ten. C. Agarose gel stained with ethidium bromide of PCR reactions using primers specific for deletion of four B. subtilis genes. Primers were designed outside of the editing template, and are therefore specific to chromosomal DNA. For all four deletions, all isolates were found to be spectinomycin sensitive, and PCR genotyping of three isolates for each deletion found that all isolates tested had the intended deletion. Note that for the control reaction for ponA , the PCR product is faint because the amplicon is approximately 5 kb and the extension time of the PCR was 3 min, which is near the limit for complete extension for Q5 DNA polymerase; the other three control amplicons are no larger than 3.4 kb.

    Article Snippet: The PCR program used to linearize pPB41 is: PCR amplify CRISPR/Cas9 using plasmid generated in step B1 ( ) Amplify plasmid generated in step B1 (pPB43 in the example) via PCR using Q5 DNA polymerase (NEB) and primers oPEB232 and oPEB234.

    Techniques: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Amplification, Mutagenesis, Plasmid Preparation

    Assay specificity for PIK 3 CA mutations. Comparison of mutant PIK 3 CA detection in human control genomic DNA ( gDNA ) and cell‐free plasma DNA following pre‐amplification or no pre‐amplification using Q5 High‐Fidelity DNA Polymerase (New England BioLabs) or TaqMan ® PreAmp Master Mix (Life Technologies). The level of blank (LoB) was 0.04% for the mutation E545K and for H1047R, 0.02% for E542K, and less than 0.01% for H1047L as shown by the graphs.

    Journal: Molecular Oncology

    Article Title: Correlation between circulating cell‐free PIK3CA tumor DNA levels and treatment response in patients with PIK3CA‐mutated metastatic breast cancer

    doi: 10.1002/1878-0261.12305

    Figure Lengend Snippet: Assay specificity for PIK 3 CA mutations. Comparison of mutant PIK 3 CA detection in human control genomic DNA ( gDNA ) and cell‐free plasma DNA following pre‐amplification or no pre‐amplification using Q5 High‐Fidelity DNA Polymerase (New England BioLabs) or TaqMan ® PreAmp Master Mix (Life Technologies). The level of blank (LoB) was 0.04% for the mutation E545K and for H1047R, 0.02% for E542K, and less than 0.01% for H1047L as shown by the graphs.

    Article Snippet: To increase the sensitivity of the analysis, the remaining 75 μL serum DNA was subjected to 12 cycles of PCR pre‐amplification with Q5 High‐Fidelity DNA Polymerase (New England BioLabs, Ipswich, MA, USA) using a multiplex PIK3CA primer mix.

    Techniques: Mutagenesis, Amplification

    Deoxynucleoside diphosphate utilization by DNA polymerases. ( A ) A standard PCR with a monophosphate substrate is inhibited, whereas the presence of all four di- or triphosphate nucleotides supports DNA amplification. Thermophilic polymerases Taq, Vent (exo−), and Pfu , as well as B , Deep Vent, and Q5 DNA polymerases, also utilize the diphosphorylated substrates. ( C and D ) Primer-extension reactions on short templates sampled at indicated time points. All reactions were performed with 100 µM dNDPs at 60 °C, except the Bsu reactions, which were performed at 37 °C. Extended sequences are shown alongside of gels and were distinct in C and D . Primer band is indicated with “–P.” Pausing appears mainly before incorporation of dA and dC and is variable among the polymerases. ( E ).

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

    Article Title: DNA synthesis from diphosphate substrates by DNA polymerases

    doi: 10.1073/pnas.1712193115

    Figure Lengend Snippet: Deoxynucleoside diphosphate utilization by DNA polymerases. ( A ) A standard PCR with a monophosphate substrate is inhibited, whereas the presence of all four di- or triphosphate nucleotides supports DNA amplification. Thermophilic polymerases Taq, Vent (exo−), and Pfu , as well as B , Deep Vent, and Q5 DNA polymerases, also utilize the diphosphorylated substrates. ( C and D ) Primer-extension reactions on short templates sampled at indicated time points. All reactions were performed with 100 µM dNDPs at 60 °C, except the Bsu reactions, which were performed at 37 °C. Extended sequences are shown alongside of gels and were distinct in C and D . Primer band is indicated with “–P.” Pausing appears mainly before incorporation of dA and dC and is variable among the polymerases. ( E ).

    Article Snippet: Taq DNA polymerase, Phusion High-Fidelity DNA polymerase, DeepVent DNA polymerase, Vent DNA polymerase, Q5 DNA High-Fidelity polymerase, Bst polymerase, Bsu DNA polymerase (large fragment), and ThermoPol Reaction Buffer [1×: 20 mM Tris⋅HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton–X–100, pH 8.8 at 25 °C] were purchased from New England Biolabs.

    Techniques: Polymerase Chain Reaction, Amplification