q5 pcr mutagenesis  (New England Biolabs)


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    Structured Review

    New England Biolabs q5 pcr mutagenesis
    Q5 Pcr Mutagenesis, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/q5 pcr mutagenesis/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    q5 pcr mutagenesis - by Bioz Stars, 2020-04
    93/100 stars

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    Related Articles

    Amplification:

    Article Title: Engineering the Genome of Thermus thermophilus Using a Counterselectable Marker
    Article Snippet: The bgaA gene was amplified from total genomic DNA from T. thermophilus IB-21 ( ). .. Q5 PCR mutagenesis was performed using a kit from New England BioLabs (catalog number E0554S).

    In Vitro:

    Article Title: Engineering the Genome of Thermus thermophilus Using a Counterselectable Marker
    Article Snippet: The pTT8 ori and repA sequences, as well as the R6Kγ ori and hph genes, were amplified from plasmid pJC1111, obtained by in vitro transposition of the hph gene as part of a synthetic transposon into pTT8 ( ). .. Q5 PCR mutagenesis was performed using a kit from New England BioLabs (catalog number E0554S).

    Synthesized:

    Article Title: Engineering the Genome of Thermus thermophilus Using a Counterselectable Marker
    Article Snippet: Oligonucleotides were synthesized by and purchased from Integrated DNA Technologies (IDT), and their sequences are listed in Tables S1 and S2 in the supplemental material. .. Q5 PCR mutagenesis was performed using a kit from New England BioLabs (catalog number E0554S).

    Mutagenesis:

    Article Title: Engineering the Genome of Thermus thermophilus Using a Counterselectable Marker
    Article Snippet: .. Q5 PCR mutagenesis was performed using a kit from New England BioLabs (catalog number E0554S). .. T. thermophilus was transformed with plasmid or genomic DNA according to the method of Koyama et al. ( ); transformants were selected on TEM plates containing 30 μg/ml kanamycin sulfate (Sigma) or 15 mM p -Cl-Phe (Sigma; catalog number C6506) and incubated at 65°C.

    Construct:

    Article Title: Engineering the Genome of Thermus thermophilus Using a Counterselectable Marker
    Article Snippet: Reporter plasmid pBGAA1 was constructed by Gibson assembly using PCR products derived from several templates as follows. .. Q5 PCR mutagenesis was performed using a kit from New England BioLabs (catalog number E0554S).

    Polymerase Chain Reaction:

    Article Title: Engineering the Genome of Thermus thermophilus Using a Counterselectable Marker
    Article Snippet: .. Q5 PCR mutagenesis was performed using a kit from New England BioLabs (catalog number E0554S). .. T. thermophilus was transformed with plasmid or genomic DNA according to the method of Koyama et al. ( ); transformants were selected on TEM plates containing 30 μg/ml kanamycin sulfate (Sigma) or 15 mM p -Cl-Phe (Sigma; catalog number C6506) and incubated at 65°C.

    Plasmid Preparation:

    Article Title: Engineering the Genome of Thermus thermophilus Using a Counterselectable Marker
    Article Snippet: The pTT8 ori and repA sequences, as well as the R6Kγ ori and hph genes, were amplified from plasmid pJC1111, obtained by in vitro transposition of the hph gene as part of a synthetic transposon into pTT8 ( ). .. Q5 PCR mutagenesis was performed using a kit from New England BioLabs (catalog number E0554S).

    Sequencing:

    Article Title: Engineering the Genome of Thermus thermophilus Using a Counterselectable Marker
    Article Snippet: The annotated sequence of pBGAA1 is presented in Fig. S9 in the supplemental material. .. Q5 PCR mutagenesis was performed using a kit from New England BioLabs (catalog number E0554S).

    Derivative Assay:

    Article Title: Engineering the Genome of Thermus thermophilus Using a Counterselectable Marker
    Article Snippet: Reporter plasmid pBGAA1 was constructed by Gibson assembly using PCR products derived from several templates as follows. .. Q5 PCR mutagenesis was performed using a kit from New England BioLabs (catalog number E0554S).

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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: 99/100, based on 782 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/q5 dna polymerase/product/New England Biolabs
    Average 99 stars, based on 782 article reviews
    Price from $9.99 to $1999.99
    q5 dna polymerase - by Bioz Stars, 2020-04
    99/100 stars
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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

    Tomato fruit colour DSB repair assay. ( a ) Crossing yellow flesh e 3756 35S:Cas9 and bicolor cc383 u6-26:Ps#1-sgRNA gives F 1 plants with a pale Bicolour fruit phenotype. F 1 plants expressing both Cas9 and gRNA were selected. The gRNA was designed for DSB induction (black lightning) in both alleles between the yellow flesh e 3756 and bicolor cc383 mutations (*). In case of error-prone NHEJ repair (blue line) of bicolor cc383 , fruit colour is yellow. In cases of non-crossover or crossover, fruit colour is expected to be red or bicolour or yellow with red spots in case of late event. Note that whole red fruits were not obtained. Rather, fruits with red spots in a yellow or bicolour background were found and are shown together with additional products of HR-induced repair in Supplementary Fig. 1 . ( b ) Fruit phenotype distribution in F 1 plants and control: Bicolour fruits are shown as orange boxes; Yellow fruits as yellow; Fruits with red sectors (putative somatic HR) are shown as red-stripped boxes. Each column represents a fruit population derived from cross of independent transgenic lines of Cas9 and a given u6-26:Ps#1-sgRNA line. The number of fruits analysed is shown on the column in black. ( c ) Sequences of the NHEJ DSB repair footprints and their relative frequency are shown. The CRISPR-Cas target sequence from the PSY1 is shown on the top. The PSY1 start codon is shown in red and the PAM in blue. The top pie represents an average of illumina Hiseq reads from 22 different F 1 plants of the cross of yellow flesh e 3756 35S:Cas9 × bicolor cc383 u6-26:Ps#1-sgRNA. The low pie represents an average of ilummina Hiseq reads from two plants of control F 1 population ( yellow flesh e 3756 × bicolor cc383 F 1 plants with no CRISPR-Cas). ( d ) Inverse PCR scheme for identification of recombinant DNA fragments (details in Supplementary Fig. 4 ). (1) DNA from separate leaves was digested with ApaI(A) and HindIII(H) and then blunted. (2) Each sample was self-ligated, and (3) amplified by two different primer sets (green and yellow). Blue- Bicolor ; red- Yellow flesh ; Dashed blue line- Bicolor deletion, *- Yellow flesh mutation. ( e ) Ratio of parental (P) versus recombinant (R) types (obtained from panel C) in individual plants. Plants 1–15- F 1 plants of the cross of yellow flesh e 3756 35S:Cas9 × bicolor cc383 u6-26:Ps#1-sgRNA; Plant 16- synthetic crossover control; Plants17–18-Yellow flesh × Bicolor (Cas9-) F 1 plants.

    Journal: Nature Communications

    Article Title: Targeted recombination between homologous chromosomes for precise breeding in tomato

    doi: 10.1038/ncomms15605

    Figure Lengend Snippet: Tomato fruit colour DSB repair assay. ( a ) Crossing yellow flesh e 3756 35S:Cas9 and bicolor cc383 u6-26:Ps#1-sgRNA gives F 1 plants with a pale Bicolour fruit phenotype. F 1 plants expressing both Cas9 and gRNA were selected. The gRNA was designed for DSB induction (black lightning) in both alleles between the yellow flesh e 3756 and bicolor cc383 mutations (*). In case of error-prone NHEJ repair (blue line) of bicolor cc383 , fruit colour is yellow. In cases of non-crossover or crossover, fruit colour is expected to be red or bicolour or yellow with red spots in case of late event. Note that whole red fruits were not obtained. Rather, fruits with red spots in a yellow or bicolour background were found and are shown together with additional products of HR-induced repair in Supplementary Fig. 1 . ( b ) Fruit phenotype distribution in F 1 plants and control: Bicolour fruits are shown as orange boxes; Yellow fruits as yellow; Fruits with red sectors (putative somatic HR) are shown as red-stripped boxes. Each column represents a fruit population derived from cross of independent transgenic lines of Cas9 and a given u6-26:Ps#1-sgRNA line. The number of fruits analysed is shown on the column in black. ( c ) Sequences of the NHEJ DSB repair footprints and their relative frequency are shown. The CRISPR-Cas target sequence from the PSY1 is shown on the top. The PSY1 start codon is shown in red and the PAM in blue. The top pie represents an average of illumina Hiseq reads from 22 different F 1 plants of the cross of yellow flesh e 3756 35S:Cas9 × bicolor cc383 u6-26:Ps#1-sgRNA. The low pie represents an average of ilummina Hiseq reads from two plants of control F 1 population ( yellow flesh e 3756 × bicolor cc383 F 1 plants with no CRISPR-Cas). ( d ) Inverse PCR scheme for identification of recombinant DNA fragments (details in Supplementary Fig. 4 ). (1) DNA from separate leaves was digested with ApaI(A) and HindIII(H) and then blunted. (2) Each sample was self-ligated, and (3) amplified by two different primer sets (green and yellow). Blue- Bicolor ; red- Yellow flesh ; Dashed blue line- Bicolor deletion, *- Yellow flesh mutation. ( e ) Ratio of parental (P) versus recombinant (R) types (obtained from panel C) in individual plants. Plants 1–15- F 1 plants of the cross of yellow flesh e 3756 35S:Cas9 × bicolor cc383 u6-26:Ps#1-sgRNA; Plant 16- synthetic crossover control; Plants17–18-Yellow flesh × Bicolor (Cas9-) F 1 plants.

    Article Snippet: High-Fidelity DNA polymerase (New England BioLabs) and 18 PCR cycles (for specific primers of each experiment see primers list).

    Techniques: Expressing, Non-Homologous End Joining, Derivative Assay, Transgenic Assay, CRISPR, Sequencing, Inverse PCR, Recombinant, Amplification, Mutagenesis