high fidelity pcr kit  (New England Biolabs)


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    Name:
    Q5 High Fidelity PCR Kit
    Description:
    Q5 High Fidelity PCR Kit 200 rxns
    Catalog Number:
    e0555l
    Price:
    327
    Size:
    200 rxns
    Category:
    PCR related Kits
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    Structured Review

    New England Biolabs high fidelity pcr kit
    Q5 High Fidelity PCR Kit
    Q5 High Fidelity PCR Kit 200 rxns
    https://www.bioz.com/result/high fidelity pcr kit/product/New England Biolabs
    Average 99 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    high fidelity pcr kit - by Bioz Stars, 2020-04
    99/100 stars

    Images

    1) Product Images from "Use of Natural Transformation To Establish an Easy Knockout Method in Riemerella anatipestifer"

    Article Title: Use of Natural Transformation To Establish an Easy Knockout Method in Riemerella anatipestifer

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00127-17

    Dependence of the transformation efficiency on the incubation time with the donor DNA. The recipient cell was R. anatipestifer ATCC 11845, and the donors were the RA0C_1193 mutagenic PCR fragments. DNase I (50 μg/ml) was added at the indicated times after the addition of DNA. Erm r transformants were selected after 1 h of incubation.
    Figure Legend Snippet: Dependence of the transformation efficiency on the incubation time with the donor DNA. The recipient cell was R. anatipestifer ATCC 11845, and the donors were the RA0C_1193 mutagenic PCR fragments. DNase I (50 μg/ml) was added at the indicated times after the addition of DNA. Erm r transformants were selected after 1 h of incubation.

    Techniques Used: Transformation Assay, Incubation, Polymerase Chain Reaction

    Transformation competition experiments. Competent R. anatipestifer ATCC 11845 was transformed with 1 μg RA0C_1193 mutagenic PCR fragments alone (control), 1 μg of RA0C_1193 mutagenic PCR fragments mixed with 1 μg of competing chromosomal DNA of E. coli XL1-Blue, or chromosomal DNA of R. anatipestifer ATCC 11845 as indicated for 1 h. The averages and standard deviations of three independent experiments are shown. The numbers above each data point represent P values from comparisons (paired one-tailed Student's t test) of the average relative transformation frequencies with E. coli DNA as the competing DNA and R. anatipestifer ATCC 11845 DNA as the competing DNA.
    Figure Legend Snippet: Transformation competition experiments. Competent R. anatipestifer ATCC 11845 was transformed with 1 μg RA0C_1193 mutagenic PCR fragments alone (control), 1 μg of RA0C_1193 mutagenic PCR fragments mixed with 1 μg of competing chromosomal DNA of E. coli XL1-Blue, or chromosomal DNA of R. anatipestifer ATCC 11845 as indicated for 1 h. The averages and standard deviations of three independent experiments are shown. The numbers above each data point represent P values from comparisons (paired one-tailed Student's t test) of the average relative transformation frequencies with E. coli DNA as the competing DNA and R. anatipestifer ATCC 11845 DNA as the competing DNA.

    Techniques Used: Transformation Assay, Polymerase Chain Reaction, One-tailed Test

    Related Articles

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    Article Snippet: Afterwards, 1 µL RNase H was added followed by an incubation step at 37 °C for 20 min. Human antibody variable regions from OmniRats® were amplified from cDNA in two successive PCR reactions using Q5 High-Fidelity 2 × Master Mix and 50 µL reaction volume (NEB). .. In the second PCR, human VH domains were amplified with primers incorporating Bsa I recognition sequences for subsequent Golden Gate Cloning.

    Centrifugation:

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    Amplification:

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    Article Snippet: .. Afterwards, 1 µL RNase H was added followed by an incubation step at 37 °C for 20 min. Human antibody variable regions from OmniRats® were amplified from cDNA in two successive PCR reactions using Q5 High-Fidelity 2 × Master Mix and 50 µL reaction volume (NEB). .. In PCR1, 12 different reactions were prepared with 5 µL cDNA using unique forward primers annealing to germline leader sequences and one reverse primer annealing to rat CH1 domain under the following conditions: 95 °C for 120 s, 30 cycles of 95 °C for 15 s, 58 °C for 30 s and 72 °C for 90 s. PCR products were purified via Wizard® SV Gel and PCR Clean-up System (Promega).

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    Article Title: Full genome characterization of porcine circovirus type 3 isolates reveals the existence of two distinct groups of virus strains
    Article Snippet: To obtain the complete PCV3 genome sequence, we amplified three overlapping PCR products (PCV374–1144 , PCV31137–1561 and PCV31427–433 ). .. We used 1 μl of the RCA reaction as template for the PCRs performed with the Q5 High Fidelity PCR Kit (New England Biolabs) using the following thermal profile: 98 °C for 5 min, and 30 cycles of 98 °C for 30 s, 55 °C for 60 s and 72 °C for 2 min. PCR products were controlled by agarose gel electrophoresis and sequenced using the PCR primers (Table ) and the sequencing service of Eurofins Genomics (Ebersberg, Germany).

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    Quantitative RT-PCR:

    Article Title: Perturbation of synapsins homeostasis through HIV-1 Tat-mediated suppression of BAG3 in primary neuronal cells
    Article Snippet: Paragraph title: Real-time quantitative RT-PCR (qRT-PCR) ... The specificity and efficiency of the primers were checked by RT-PCR using Q5 High-Fidelity PCR Kit (New England Biolabs).

    Adsorption:

    Article Title: Zika Virus Encoding Nonglycosylated Envelope Protein Is Attenuated and Defective in Neuroinvasion
    Article Snippet: Following virus adsorption, the inoculum was removed, and the cell monolayers were overlaid with medium containing 1% low-gelling-temperature (LGT) agarose in VGM. .. Restriction enzymes, DNA-modifying enzymes, the Q5 High Fidelity PCR kit, the ProtoScript II First Strand cDNA synthesis kit, endo H, and PNGase F were obtained from New England BioLabs (Ipswich, MA).

    SYBR Green Assay:

    Article Title: Perturbation of synapsins homeostasis through HIV-1 Tat-mediated suppression of BAG3 in primary neuronal cells
    Article Snippet: The specificity and efficiency of the primers were checked by RT-PCR using Q5 High-Fidelity PCR Kit (New England Biolabs). .. All qPCR reactions were performed with the LightCycler96® (Roche) using the SYBR™ Green master mix (Applied Biosystems, ThermoFisher), according to manufacturer’s protocol.

    Random Hexamer Labeling:

    Article Title: A novel one-step approach for the construction of yeast surface display Fab antibody libraries
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    Cell Culture:

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    Expressing:

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    Modification:

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    Article Snippet: Therefore, we modified the primer setup initially described by Paliski and colleagues [ ] (see Table ). .. We used 1 μl of the RCA reaction as template for the PCRs performed with the Q5 High Fidelity PCR Kit (New England Biolabs) using the following thermal profile: 98 °C for 5 min, and 30 cycles of 98 °C for 30 s, 55 °C for 60 s and 72 °C for 2 min. PCR products were controlled by agarose gel electrophoresis and sequenced using the PCR primers (Table ) and the sequencing service of Eurofins Genomics (Ebersberg, Germany).

    Transfection:

    Article Title: NLRP11 attenuates Toll-like receptor signalling by targeting TRAF6 for degradation via the ubiquitin ligase RNF19A
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    Sequencing:

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    Article Title: Full genome characterization of porcine circovirus type 3 isolates reveals the existence of two distinct groups of virus strains
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    Incubation:

    Article Title: A novel one-step approach for the construction of yeast surface display Fab antibody libraries
    Article Snippet: .. Afterwards, 1 µL RNase H was added followed by an incubation step at 37 °C for 20 min. Human antibody variable regions from OmniRats® were amplified from cDNA in two successive PCR reactions using Q5 High-Fidelity 2 × Master Mix and 50 µL reaction volume (NEB). .. In PCR1, 12 different reactions were prepared with 5 µL cDNA using unique forward primers annealing to germline leader sequences and one reverse primer annealing to rat CH1 domain under the following conditions: 95 °C for 120 s, 30 cycles of 95 °C for 15 s, 58 °C for 30 s and 72 °C for 90 s. PCR products were purified via Wizard® SV Gel and PCR Clean-up System (Promega).

    Article Title: Characterization of the total and viable bacterial and fungal communities associated with the International Space Station surfaces
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    Article Title: Zika Virus Encoding Nonglycosylated Envelope Protein Is Attenuated and Defective in Neuroinvasion
    Article Snippet: After incubation for 5 days at 37°C, the cells were fixed in 10% formaldehyde in PBS for 30 min, the agarose plugs were removed, and the monolayers were stained with 0.1% crystal violet in 30% methanol. .. Restriction enzymes, DNA-modifying enzymes, the Q5 High Fidelity PCR kit, the ProtoScript II First Strand cDNA synthesis kit, endo H, and PNGase F were obtained from New England BioLabs (Ipswich, MA).

    Infection:

    Article Title: Zika Virus Encoding Nonglycosylated Envelope Protein Is Attenuated and Defective in Neuroinvasion
    Article Snippet: Small aliquots of infected cell culture supernatants were collected at various times postinfection, clarified, and stored at −80°C for virus titration at a later time. .. Restriction enzymes, DNA-modifying enzymes, the Q5 High Fidelity PCR kit, the ProtoScript II First Strand cDNA synthesis kit, endo H, and PNGase F were obtained from New England BioLabs (Ipswich, MA).

    Generated:

    Article Title: First identification of porcine parvovirus 6 in Poland
    Article Snippet: The generated standard curve with a regression value of R 2 = 0.989 was linear and showed a reaction efficiency of 114.092. .. The amplifications were performed using the Q5 High-Fidelity PCR kit (NEB) in a 50 µl volume containing 25 µl of Q5 High-Fidelity 2× mix, 2 µl of a 10 µM forward primer, 2 µl of a 10 µM reverse primer, 2 µl of the extracted DNA, and 19 µl of ddH2 O under the following thermocycler conditions: denaturation for 30 s at 98 °C followed by 30 cycles of 98 °C for 10 s, 55 °C for 30 s, and 72 °C for 60 s, and a final extension step at 72 °C for 10 min.

    Article Title: Use of Natural Transformation To Establish an Easy Knockout Method in Riemerella anatipestifer
    Article Snippet: Small-scale plasmid DNA preparations were generated using a Tianprep mini plasmid kit (Tiangen, Beijing, China). .. The DNA fragments were amplified in a Hybaid PCR thermocycler using the high-fidelity PCR kit (NEB, USA).

    Polymerase Chain Reaction:

    Article Title: A novel one-step approach for the construction of yeast surface display Fab antibody libraries
    Article Snippet: .. Afterwards, 1 µL RNase H was added followed by an incubation step at 37 °C for 20 min. Human antibody variable regions from OmniRats® were amplified from cDNA in two successive PCR reactions using Q5 High-Fidelity 2 × Master Mix and 50 µL reaction volume (NEB). .. In PCR1, 12 different reactions were prepared with 5 µL cDNA using unique forward primers annealing to germline leader sequences and one reverse primer annealing to rat CH1 domain under the following conditions: 95 °C for 120 s, 30 cycles of 95 °C for 15 s, 58 °C for 30 s and 72 °C for 90 s. PCR products were purified via Wizard® SV Gel and PCR Clean-up System (Promega).

    Article Title: Characterization of the total and viable bacterial and fungal communities associated with the International Space Station surfaces
    Article Snippet: .. PCR synthesis of SSU-V4 and ITS1 amplicons was performed using Q5 High-Fidelity PCR Kit (New England Biolabs, Ipswich, MA) according to the manufacturer’s instructions. .. The 40-μL reaction mixtures were incubated under the following conditions: initial denaturation at 94 °C for 3 min followed by 35 cycles of 94 °C for 30 s, 47 °C for 30 s, and 72 °C for 90 s, with a final extension at 72 °C for 5 min.

    Article Title: Perturbation of synapsins homeostasis through HIV-1 Tat-mediated suppression of BAG3 in primary neuronal cells
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    Article Title: First identification of porcine parvovirus 6 in Poland
    Article Snippet: .. The amplifications were performed using the Q5 High-Fidelity PCR kit (NEB) in a 50 µl volume containing 25 µl of Q5 High-Fidelity 2× mix, 2 µl of a 10 µM forward primer, 2 µl of a 10 µM reverse primer, 2 µl of the extracted DNA, and 19 µl of ddH2 O under the following thermocycler conditions: denaturation for 30 s at 98 °C followed by 30 cycles of 98 °C for 10 s, 55 °C for 30 s, and 72 °C for 60 s, and a final extension step at 72 °C for 10 min. .. The PCR products were purified by the PureLink™ Quick Gel Extraction and PCR Purification Combo Kit (Invitrogen, Life Technologies) according to the manufacturer’s instructions and were sequenced at the Edinburgh Genomics facility, University of Edinburgh, Edinburgh, United Kingdom.

    Article Title: An Omics Approach to Extracellular Vesicles from HIV-1 Infected Cells
    Article Snippet: .. PCR was performed using Q5 High-Fidelity PCR Kit (New England Biolabs) under the following conditions: 98 °C for 30 s; 35 cycles of 98 °C for 10 s, 62 °C for 30 s, and 72 °C for 30 s; and 72 °C for 2 min. ..

    Article Title: Spring flowering habit in field pennycress (Thlaspi arvense) has arisen multiple independent times. Spring flowering habit in field pennycress (Thlaspi arvense) has arisen multiple independent times
    Article Snippet: .. New England Biolabs Q5 High‐Fidelity PCR Kit with 2× Master Mix was used, with the following thermal cycler conditions: (a) 98 ° C for 30 s, (b) 98 ° C for 10 s, (c) 57 ° C for 20 s, (d) 72 ° C for 20 s, (e) Go to step #2 34 times, (f) 72 ° C for 2 min, (g) 4 ° C hold. ..

    Article Title: Use of Natural Transformation To Establish an Easy Knockout Method in Riemerella anatipestifer
    Article Snippet: .. The DNA fragments were amplified in a Hybaid PCR thermocycler using the high-fidelity PCR kit (NEB, USA). .. Purification of DNA fragments from the PCR and the restriction reaction was performed using the Tiangen extract II kit (Tiangen, Beijing, China).

    Article Title: Targeted deletion of the zebrafish actin-bundling protein L-plastin (lcp1)
    Article Snippet: .. This specific forward was then matched with a generic reverse for high-fidelity PCR amplification (Q5 PCR Kit, New England BioLabs) from a publicly available gRNA template (pT7-gRNA, Addgene #46759) [ ]. .. The resulting 120-bp amplicon was cleaned by phenol-chloroform extraction, and then precipitated by addition of 100% ethanol, 3M sodium acetate, and 1 μL GlycoBlueTM .

    Article Title: Full genome characterization of porcine circovirus type 3 isolates reveals the existence of two distinct groups of virus strains
    Article Snippet: .. We used 1 μl of the RCA reaction as template for the PCRs performed with the Q5 High Fidelity PCR Kit (New England Biolabs) using the following thermal profile: 98 °C for 5 min, and 30 cycles of 98 °C for 30 s, 55 °C for 60 s and 72 °C for 2 min. PCR products were controlled by agarose gel electrophoresis and sequenced using the PCR primers (Table ) and the sequencing service of Eurofins Genomics (Ebersberg, Germany). .. Sequence assembly and analysis DNASTAR Lasergene and MEGA6 software was used for assembly, alignment and analysis of the sequences.

    Article Title: NLRP11 attenuates Toll-like receptor signalling by targeting TRAF6 for degradation via the ubiquitin ligase RNF19A
    Article Snippet: .. Site-directed mutagenesis The template plasmid was amplified with a pair of primers containing a point mutation by means of the Q5 High-Fidelity PCR Kit (New England Biolabs), and the products were subsequently digested with 10 U of DpnI (New England Biolabs) at 37 °C for 1 h and were transfected into DH5α competent cells. ..

    Article Title: Zika Virus Encoding Nonglycosylated Envelope Protein Is Attenuated and Defective in Neuroinvasion
    Article Snippet: .. Restriction enzymes, DNA-modifying enzymes, the Q5 High Fidelity PCR kit, the ProtoScript II First Strand cDNA synthesis kit, endo H, and PNGase F were obtained from New England BioLabs (Ipswich, MA). .. SuperScript II was obtained from Invitrogen (Carlsbad, CA).

    Binding Assay:

    Article Title: Targeted deletion of the zebrafish actin-bundling protein L-plastin (lcp1)
    Article Snippet: Template assembly and in vitro transcription of guide RNA A gRNA forward primer was designed such that an upstream SP6 polymerase binding site flanked the desired lcp1 exon 2 target sequence ( , Oligos and Primers). .. This specific forward was then matched with a generic reverse for high-fidelity PCR amplification (Q5 PCR Kit, New England BioLabs) from a publicly available gRNA template (pT7-gRNA, Addgene #46759) [ ].

    Plaque Assay:

    Article Title: Zika Virus Encoding Nonglycosylated Envelope Protein Is Attenuated and Defective in Neuroinvasion
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    Mutagenesis:

    Article Title: Spring flowering habit in field pennycress (Thlaspi arvense) has arisen multiple independent times. Spring flowering habit in field pennycress (Thlaspi arvense) has arisen multiple independent times
    Article Snippet: Paragraph title: Analysis of FLC mutations in MN111 × MN108SA F2 population, global spring varieties, and EMS mutant ... New England Biolabs Q5 High‐Fidelity PCR Kit with 2× Master Mix was used, with the following thermal cycler conditions: (a) 98 ° C for 30 s, (b) 98 ° C for 10 s, (c) 57 ° C for 20 s, (d) 72 ° C for 20 s, (e) Go to step #2 34 times, (f) 72 ° C for 2 min, (g) 4 ° C hold.

    Article Title: NLRP11 attenuates Toll-like receptor signalling by targeting TRAF6 for degradation via the ubiquitin ligase RNF19A
    Article Snippet: .. Site-directed mutagenesis The template plasmid was amplified with a pair of primers containing a point mutation by means of the Q5 High-Fidelity PCR Kit (New England Biolabs), and the products were subsequently digested with 10 U of DpnI (New England Biolabs) at 37 °C for 1 h and were transfected into DH5α competent cells. ..

    Isolation:

    Article Title: Characterization of the total and viable bacterial and fungal communities associated with the International Space Station surfaces
    Article Snippet: PCR synthesis of SSU-V4 and ITS1 amplicons was performed using Q5 High-Fidelity PCR Kit (New England Biolabs, Ipswich, MA) according to the manufacturer’s instructions. .. The amplicons were isolated from gel slices using silica spin-columns [ ], and eluted with nano-pure water.

    Article Title: Spring flowering habit in field pennycress (Thlaspi arvense) has arisen multiple independent times. Spring flowering habit in field pennycress (Thlaspi arvense) has arisen multiple independent times
    Article Snippet: 2.4 Analysis of FLC mutations in MN111 × MN108SA F2 population, global spring varieties, and EMS mutant DNA was isolated from F2 progeny grown in the conditions described above using the Omega Bio‐Tek Plant MagBind 96 kit according to the manufacturers recommended protocol. .. New England Biolabs Q5 High‐Fidelity PCR Kit with 2× Master Mix was used, with the following thermal cycler conditions: (a) 98 ° C for 30 s, (b) 98 ° C for 10 s, (c) 57 ° C for 20 s, (d) 72 ° C for 20 s, (e) Go to step #2 34 times, (f) 72 ° C for 2 min, (g) 4 ° C hold.

    Article Title: Full genome characterization of porcine circovirus type 3 isolates reveals the existence of two distinct groups of virus strains
    Article Snippet: Briefly, 1 μl of isolated DNA was mixed together with 5 μl of TempliPhi sample buffer. .. We used 1 μl of the RCA reaction as template for the PCRs performed with the Q5 High Fidelity PCR Kit (New England Biolabs) using the following thermal profile: 98 °C for 5 min, and 30 cycles of 98 °C for 30 s, 55 °C for 60 s and 72 °C for 2 min. PCR products were controlled by agarose gel electrophoresis and sequenced using the PCR primers (Table ) and the sequencing service of Eurofins Genomics (Ebersberg, Germany).

    Purification:

    Article Title: A novel one-step approach for the construction of yeast surface display Fab antibody libraries
    Article Snippet: Afterwards, 1 µL RNase H was added followed by an incubation step at 37 °C for 20 min. Human antibody variable regions from OmniRats® were amplified from cDNA in two successive PCR reactions using Q5 High-Fidelity 2 × Master Mix and 50 µL reaction volume (NEB). .. In PCR1, 12 different reactions were prepared with 5 µL cDNA using unique forward primers annealing to germline leader sequences and one reverse primer annealing to rat CH1 domain under the following conditions: 95 °C for 120 s, 30 cycles of 95 °C for 15 s, 58 °C for 30 s and 72 °C for 90 s. PCR products were purified via Wizard® SV Gel and PCR Clean-up System (Promega).

    Article Title: First identification of porcine parvovirus 6 in Poland
    Article Snippet: The amplifications were performed using the Q5 High-Fidelity PCR kit (NEB) in a 50 µl volume containing 25 µl of Q5 High-Fidelity 2× mix, 2 µl of a 10 µM forward primer, 2 µl of a 10 µM reverse primer, 2 µl of the extracted DNA, and 19 µl of ddH2 O under the following thermocycler conditions: denaturation for 30 s at 98 °C followed by 30 cycles of 98 °C for 10 s, 55 °C for 30 s, and 72 °C for 60 s, and a final extension step at 72 °C for 10 min. .. The PCR products were purified by the PureLink™ Quick Gel Extraction and PCR Purification Combo Kit (Invitrogen, Life Technologies) according to the manufacturer’s instructions and were sequenced at the Edinburgh Genomics facility, University of Edinburgh, Edinburgh, United Kingdom.

    Article Title: Spring flowering habit in field pennycress (Thlaspi arvense) has arisen multiple independent times. Spring flowering habit in field pennycress (Thlaspi arvense) has arisen multiple independent times
    Article Snippet: New England Biolabs Q5 High‐Fidelity PCR Kit with 2× Master Mix was used, with the following thermal cycler conditions: (a) 98 ° C for 30 s, (b) 98 ° C for 10 s, (c) 57 ° C for 20 s, (d) 72 ° C for 20 s, (e) Go to step #2 34 times, (f) 72 ° C for 2 min, (g) 4 ° C hold. .. PCR products were submitted to Beckman Coulter Genomics for PCR product purification and single pass Sanger sequencing.

    Article Title: Use of Natural Transformation To Establish an Easy Knockout Method in Riemerella anatipestifer
    Article Snippet: The DNA fragments were amplified in a Hybaid PCR thermocycler using the high-fidelity PCR kit (NEB, USA). .. Purification of DNA fragments from the PCR and the restriction reaction was performed using the Tiangen extract II kit (Tiangen, Beijing, China).

    Article Title: Targeted deletion of the zebrafish actin-bundling protein L-plastin (lcp1)
    Article Snippet: This specific forward was then matched with a generic reverse for high-fidelity PCR amplification (Q5 PCR Kit, New England BioLabs) from a publicly available gRNA template (pT7-gRNA, Addgene #46759) [ ]. .. After vigorous centrifugation (20,000 g for 15 min) the purified pellet was air-dried and resuspended in 20 uL of nuclease-free water.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Perturbation of synapsins homeostasis through HIV-1 Tat-mediated suppression of BAG3 in primary neuronal cells
    Article Snippet: .. The specificity and efficiency of the primers were checked by RT-PCR using Q5 High-Fidelity PCR Kit (New England Biolabs). .. All qPCR reactions were performed with the LightCycler96® (Roche) using the SYBR™ Green master mix (Applied Biosystems, ThermoFisher), according to manufacturer’s protocol.

    Gel Extraction:

    Article Title: First identification of porcine parvovirus 6 in Poland
    Article Snippet: The amplifications were performed using the Q5 High-Fidelity PCR kit (NEB) in a 50 µl volume containing 25 µl of Q5 High-Fidelity 2× mix, 2 µl of a 10 µM forward primer, 2 µl of a 10 µM reverse primer, 2 µl of the extracted DNA, and 19 µl of ddH2 O under the following thermocycler conditions: denaturation for 30 s at 98 °C followed by 30 cycles of 98 °C for 10 s, 55 °C for 30 s, and 72 °C for 60 s, and a final extension step at 72 °C for 10 min. .. The PCR products were purified by the PureLink™ Quick Gel Extraction and PCR Purification Combo Kit (Invitrogen, Life Technologies) according to the manufacturer’s instructions and were sequenced at the Edinburgh Genomics facility, University of Edinburgh, Edinburgh, United Kingdom.

    Titration:

    Article Title: Zika Virus Encoding Nonglycosylated Envelope Protein Is Attenuated and Defective in Neuroinvasion
    Article Snippet: Small aliquots of infected cell culture supernatants were collected at various times postinfection, clarified, and stored at −80°C for virus titration at a later time. .. Restriction enzymes, DNA-modifying enzymes, the Q5 High Fidelity PCR kit, the ProtoScript II First Strand cDNA synthesis kit, endo H, and PNGase F were obtained from New England BioLabs (Ipswich, MA).

    Plasmid Preparation:

    Article Title: Use of Natural Transformation To Establish an Easy Knockout Method in Riemerella anatipestifer
    Article Snippet: Small-scale plasmid DNA preparations were generated using a Tianprep mini plasmid kit (Tiangen, Beijing, China). .. The DNA fragments were amplified in a Hybaid PCR thermocycler using the high-fidelity PCR kit (NEB, USA).

    Article Title: NLRP11 attenuates Toll-like receptor signalling by targeting TRAF6 for degradation via the ubiquitin ligase RNF19A
    Article Snippet: .. Site-directed mutagenesis The template plasmid was amplified with a pair of primers containing a point mutation by means of the Q5 High-Fidelity PCR Kit (New England Biolabs), and the products were subsequently digested with 10 U of DpnI (New England Biolabs) at 37 °C for 1 h and were transfected into DH5α competent cells. ..

    Software:

    Article Title: First identification of porcine parvovirus 6 in Poland
    Article Snippet: The amplifications were performed using the Q5 High-Fidelity PCR kit (NEB) in a 50 µl volume containing 25 µl of Q5 High-Fidelity 2× mix, 2 µl of a 10 µM forward primer, 2 µl of a 10 µM reverse primer, 2 µl of the extracted DNA, and 19 µl of ddH2 O under the following thermocycler conditions: denaturation for 30 s at 98 °C followed by 30 cycles of 98 °C for 10 s, 55 °C for 30 s, and 72 °C for 60 s, and a final extension step at 72 °C for 10 min. .. The sequences were assembled by the SeqMan program of Lasergene 7.0 software (DNASTAR Inc., Madison, Wisconsin, USA) and compared with other available PPV6 sequences using the BLAST web-based program ( http://www.ncbi.nlm.nih.gov/BLAST ).

    Real-time Polymerase Chain Reaction:

    Article Title: Perturbation of synapsins homeostasis through HIV-1 Tat-mediated suppression of BAG3 in primary neuronal cells
    Article Snippet: The specificity and efficiency of the primers were checked by RT-PCR using Q5 High-Fidelity PCR Kit (New England Biolabs). .. All qPCR reactions were performed with the LightCycler96® (Roche) using the SYBR™ Green master mix (Applied Biosystems, ThermoFisher), according to manufacturer’s protocol.

    Article Title: First identification of porcine parvovirus 6 in Poland
    Article Snippet: The sensitivity of the PPV6 real-time PCR assay was determined by testing 10-fold serial dilutions of the PPV6 DNA standard in triplicate. .. The amplifications were performed using the Q5 High-Fidelity PCR kit (NEB) in a 50 µl volume containing 25 µl of Q5 High-Fidelity 2× mix, 2 µl of a 10 µM forward primer, 2 µl of a 10 µM reverse primer, 2 µl of the extracted DNA, and 19 µl of ddH2 O under the following thermocycler conditions: denaturation for 30 s at 98 °C followed by 30 cycles of 98 °C for 10 s, 55 °C for 30 s, and 72 °C for 60 s, and a final extension step at 72 °C for 10 min.

    Agarose Gel Electrophoresis:

    Article Title: Full genome characterization of porcine circovirus type 3 isolates reveals the existence of two distinct groups of virus strains
    Article Snippet: .. We used 1 μl of the RCA reaction as template for the PCRs performed with the Q5 High Fidelity PCR Kit (New England Biolabs) using the following thermal profile: 98 °C for 5 min, and 30 cycles of 98 °C for 30 s, 55 °C for 60 s and 72 °C for 2 min. PCR products were controlled by agarose gel electrophoresis and sequenced using the PCR primers (Table ) and the sequencing service of Eurofins Genomics (Ebersberg, Germany). .. Sequence assembly and analysis DNASTAR Lasergene and MEGA6 software was used for assembly, alignment and analysis of the sequences.

    In Vitro:

    Article Title: Targeted deletion of the zebrafish actin-bundling protein L-plastin (lcp1)
    Article Snippet: Paragraph title: Template assembly and in vitro transcription of guide RNA ... This specific forward was then matched with a generic reverse for high-fidelity PCR amplification (Q5 PCR Kit, New England BioLabs) from a publicly available gRNA template (pT7-gRNA, Addgene #46759) [ ].

    Quantitation Assay:

    Article Title: Zika Virus Encoding Nonglycosylated Envelope Protein Is Attenuated and Defective in Neuroinvasion
    Article Snippet: Virus quantitation was performed by plaque assay on Vero cells. .. Restriction enzymes, DNA-modifying enzymes, the Q5 High Fidelity PCR kit, the ProtoScript II First Strand cDNA synthesis kit, endo H, and PNGase F were obtained from New England BioLabs (Ipswich, MA).

    Sampling:

    Article Title: Characterization of the total and viable bacterial and fungal communities associated with the International Space Station surfaces
    Article Snippet: Paragraph title: Flight sampling 1 and 2 ... PCR synthesis of SSU-V4 and ITS1 amplicons was performed using Q5 High-Fidelity PCR Kit (New England Biolabs, Ipswich, MA) according to the manufacturer’s instructions.

    Staining:

    Article Title: Zika Virus Encoding Nonglycosylated Envelope Protein Is Attenuated and Defective in Neuroinvasion
    Article Snippet: After incubation for 5 days at 37°C, the cells were fixed in 10% formaldehyde in PBS for 30 min, the agarose plugs were removed, and the monolayers were stained with 0.1% crystal violet in 30% methanol. .. Restriction enzymes, DNA-modifying enzymes, the Q5 High Fidelity PCR kit, the ProtoScript II First Strand cDNA synthesis kit, endo H, and PNGase F were obtained from New England BioLabs (Ipswich, MA).

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    New England Biolabs high fidelity pcr kit
    Dependence of the transformation efficiency on the incubation time with the donor <t>DNA.</t> The recipient cell was R. anatipestifer ATCC 11845, and the donors were the RA0C_1193 mutagenic <t>PCR</t> fragments. DNase I (50 μg/ml) was added at the indicated times after the addition of DNA. Erm r transformants were selected after 1 h of incubation.
    High Fidelity Pcr Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Dependence of the transformation efficiency on the incubation time with the donor DNA. The recipient cell was R. anatipestifer ATCC 11845, and the donors were the RA0C_1193 mutagenic PCR fragments. DNase I (50 μg/ml) was added at the indicated times after the addition of DNA. Erm r transformants were selected after 1 h of incubation.

    Journal: Applied and Environmental Microbiology

    Article Title: Use of Natural Transformation To Establish an Easy Knockout Method in Riemerella anatipestifer

    doi: 10.1128/AEM.00127-17

    Figure Lengend Snippet: Dependence of the transformation efficiency on the incubation time with the donor DNA. The recipient cell was R. anatipestifer ATCC 11845, and the donors were the RA0C_1193 mutagenic PCR fragments. DNase I (50 μg/ml) was added at the indicated times after the addition of DNA. Erm r transformants were selected after 1 h of incubation.

    Article Snippet: The DNA fragments were amplified in a Hybaid PCR thermocycler using the high-fidelity PCR kit (NEB, USA).

    Techniques: Transformation Assay, Incubation, Polymerase Chain Reaction

    Transformation competition experiments. Competent R. anatipestifer ATCC 11845 was transformed with 1 μg RA0C_1193 mutagenic PCR fragments alone (control), 1 μg of RA0C_1193 mutagenic PCR fragments mixed with 1 μg of competing chromosomal DNA of E. coli XL1-Blue, or chromosomal DNA of R. anatipestifer ATCC 11845 as indicated for 1 h. The averages and standard deviations of three independent experiments are shown. The numbers above each data point represent P values from comparisons (paired one-tailed Student's t test) of the average relative transformation frequencies with E. coli DNA as the competing DNA and R. anatipestifer ATCC 11845 DNA as the competing DNA.

    Journal: Applied and Environmental Microbiology

    Article Title: Use of Natural Transformation To Establish an Easy Knockout Method in Riemerella anatipestifer

    doi: 10.1128/AEM.00127-17

    Figure Lengend Snippet: Transformation competition experiments. Competent R. anatipestifer ATCC 11845 was transformed with 1 μg RA0C_1193 mutagenic PCR fragments alone (control), 1 μg of RA0C_1193 mutagenic PCR fragments mixed with 1 μg of competing chromosomal DNA of E. coli XL1-Blue, or chromosomal DNA of R. anatipestifer ATCC 11845 as indicated for 1 h. The averages and standard deviations of three independent experiments are shown. The numbers above each data point represent P values from comparisons (paired one-tailed Student's t test) of the average relative transformation frequencies with E. coli DNA as the competing DNA and R. anatipestifer ATCC 11845 DNA as the competing DNA.

    Article Snippet: The DNA fragments were amplified in a Hybaid PCR thermocycler using the high-fidelity PCR kit (NEB, USA).

    Techniques: Transformation Assay, Polymerase Chain Reaction, One-tailed Test