phusion master mix  (New England Biolabs)


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    Name:
    Phusion High Fidelity PCR Master Mix with GC Buffer
    Description:
    Phusion High Fidelity PCR Master Mix with GC Buffer 500 rxns 50 ul vol
    Catalog Number:
    m0532l
    Price:
    736
    Size:
    500 rxns
    Category:
    Thermostable DNA Polymerases
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    Structured Review

    New England Biolabs phusion master mix
    Phusion High Fidelity PCR Master Mix with GC Buffer
    Phusion High Fidelity PCR Master Mix with GC Buffer 500 rxns 50 ul vol
    https://www.bioz.com/result/phusion master mix/product/New England Biolabs
    Average 99 stars, based on 180 article reviews
    Price from $9.99 to $1999.99
    phusion master mix - by Bioz Stars, 2020-08
    99/100 stars

    Images

    1) Product Images from "Improved PCR Amplification of Broad Spectrum GC DNA Templates"

    Article Title: Improved PCR Amplification of Broad Spectrum GC DNA Templates

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0156478

    Schematics of the subcycling PCR protocol. A. Each PCR cycle involves a denaturation step and an annealing/elongation step. We introduced 4X sub-cycling the annealing/elongation step within each of the 30X amplification cycles. B. Multiplexed amplification products for pools of 7 oligos with 154–200 bp length products of varying GC content. Phusion and KAPA HIFI polymerases were used with and without a sub-cycling thermocycle. Each different condition is used to amplify 12 separate oligo pools with GC content ranges as follows: 1.) 16.4–34.3; 2.) 13.5–38.7; 3.) 21.5–37.3; 4.) 12.2–12.2; 5.) 12.7–40.0; 6.) 14.9–41.6; 7.) 16.4–37.6; 8.) 12.7–42.0; 9.) 20.9–40.6; 10.) 12.5–42.5; 11.) 12.7–43.7; 12.) 14.8–35.6. Results are based on electronic gels created by electrophoresis using a Perkin Elmer GX instrument with a 5k chip. *Bin shows an example of an expected PCR pattern where a strong 154-200bp product band is seen. PCR reactions were not purified and primers can be seen at the bottom of each sample.
    Figure Legend Snippet: Schematics of the subcycling PCR protocol. A. Each PCR cycle involves a denaturation step and an annealing/elongation step. We introduced 4X sub-cycling the annealing/elongation step within each of the 30X amplification cycles. B. Multiplexed amplification products for pools of 7 oligos with 154–200 bp length products of varying GC content. Phusion and KAPA HIFI polymerases were used with and without a sub-cycling thermocycle. Each different condition is used to amplify 12 separate oligo pools with GC content ranges as follows: 1.) 16.4–34.3; 2.) 13.5–38.7; 3.) 21.5–37.3; 4.) 12.2–12.2; 5.) 12.7–40.0; 6.) 14.9–41.6; 7.) 16.4–37.6; 8.) 12.7–42.0; 9.) 20.9–40.6; 10.) 12.5–42.5; 11.) 12.7–43.7; 12.) 14.8–35.6. Results are based on electronic gels created by electrophoresis using a Perkin Elmer GX instrument with a 5k chip. *Bin shows an example of an expected PCR pattern where a strong 154-200bp product band is seen. PCR reactions were not purified and primers can be seen at the bottom of each sample.

    Techniques Used: Polymerase Chain Reaction, Amplification, Electrophoresis, Chromatin Immunoprecipitation, Purification

    Related Articles

    Amplification:

    Article Title: The Tsetse Fly Displays an Attenuated Immune Response to Its Secondary Symbiont, Sodalis glossinidius
    Article Snippet: .. RNAi-Mediated Imd-Pathway Suppression To construct the relish dsRNA, the complete relish coding sequence (2,599 bp) was first PCR amplified using the Phusion High-Fidelity PCR master mix (New England BioLabs) in a total volume of 50 μl and 0.5 μM of each primer ( ) with following cycling conditions: 30 sec./98°C, 35 cycles 10 sec./98°C, 30 sec./65°C, 30 sec./72°C, and 10 min/72°C. .. For this, cDNA was generated from total RNA isolated from 10-day old wild-type abdomens as described above.

    Article Title: Amplification of the MET receptor drives resistance to anti-EGFR therapies in colorectal cancer
    Article Snippet: .. The first amplification was performed in a 50 μl PCR reaction, 1× Phusion high-fidelity buffer, 1.5 U Hotstart Phusion polymerase (NEB, BioLabs), 0.5 μM of each primer with tag sequence, 0.2 mM of each deoxynucleoside triphosphate, and 0.5 mM MgCl2. .. Amplification was carried out using the following cycling conditions: 98°C for 45 s; 2 cycles of 98 °C for 10 s, 67°C for 10 s, 72°C for 10 s; 2 cycles of 98°C for 10 s, 64°C for 10 s, 72°C for 10 s; 2 cycles of 98°C for 10 s, 61°C for 10 s, 72°C for 10 s; 31 cycles of 98°C for 10 s, 58°C for 10 s, 72°C for 10 s. PCR products were diluted, and quantified using the PicoGreen double-stranded DNA assay (Invitrogen).

    Radial Immuno Diffusion:

    Article Title: The Tsetse Fly Displays an Attenuated Immune Response to Its Secondary Symbiont, Sodalis glossinidius
    Article Snippet: .. RNAi-Mediated Imd-Pathway Suppression To construct the relish dsRNA, the complete relish coding sequence (2,599 bp) was first PCR amplified using the Phusion High-Fidelity PCR master mix (New England BioLabs) in a total volume of 50 μl and 0.5 μM of each primer ( ) with following cycling conditions: 30 sec./98°C, 35 cycles 10 sec./98°C, 30 sec./65°C, 30 sec./72°C, and 10 min/72°C. .. For this, cDNA was generated from total RNA isolated from 10-day old wild-type abdomens as described above.

    Polymerase Chain Reaction:

    Article Title: An Assessment of Heavy Ion Irradiation Mutagenesis for Reverse Genetics in Wheat (Triticum aestivum L.)
    Article Snippet: .. PCR master mix stock composed of 5 µL High Fidelity Phusion PCR Buffer (New England BioLabs, Inc.), 0.5 µL 10 mM dNTPs, 0.75 µL DMSO, 0.25 µL Phusion proofreading DNA polymerase and 15.5 µL DNase-free water per 22 µL was prepared in a 2.0 mL safe-lock PCR tube. .. An Eppendorf EpMotion 5070 (Eppendorf AG, Hamburg, Germany) was used to combine 1.5 µL of a specific primer pair, 1.5 µL of 100 µM DNA and 22 µL PCR master mix into individual wells of a new half-skirt 96-well PCR plates (PCR-96M2-HS; Axygen, USA).

    Article Title: The Tsetse Fly Displays an Attenuated Immune Response to Its Secondary Symbiont, Sodalis glossinidius
    Article Snippet: .. RNAi-Mediated Imd-Pathway Suppression To construct the relish dsRNA, the complete relish coding sequence (2,599 bp) was first PCR amplified using the Phusion High-Fidelity PCR master mix (New England BioLabs) in a total volume of 50 μl and 0.5 μM of each primer ( ) with following cycling conditions: 30 sec./98°C, 35 cycles 10 sec./98°C, 30 sec./65°C, 30 sec./72°C, and 10 min/72°C. .. For this, cDNA was generated from total RNA isolated from 10-day old wild-type abdomens as described above.

    Article Title: Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries
    Article Snippet: .. Standard reactions contained 1× Phusion High-Fidelity PCR master mix with HF buffer (NEB). .. Standard (short denaturation) thermocycling for PCR with Phusion was 30 s at 98°C for the initial denaturation followed by 10 cycles of 10 s at 98°C, 30 s at 65°C and 30 s at 72°C and a final extension for 5 minutes at 72°C.

    Article Title: High-throughput sequence analysis reveals variation in the relative abundance of components of the bacterial and fungal microbiota in the rhizosphere of Ginkgo biloba
    Article Snippet: .. Each 30 µl PCR reaction mixture contained 5∼10 ng DNA template, 15 µl 2 × Master Mix (Phusion® High-Fidelity PCR Master Mix with GC Buffer, New England Biolabs, USA), with each primer in the reaction mixture being supplied at a concentration of 3 µM. ..

    Article Title: Amplification of the MET receptor drives resistance to anti-EGFR therapies in colorectal cancer
    Article Snippet: .. The first amplification was performed in a 50 μl PCR reaction, 1× Phusion high-fidelity buffer, 1.5 U Hotstart Phusion polymerase (NEB, BioLabs), 0.5 μM of each primer with tag sequence, 0.2 mM of each deoxynucleoside triphosphate, and 0.5 mM MgCl2. .. Amplification was carried out using the following cycling conditions: 98°C for 45 s; 2 cycles of 98 °C for 10 s, 67°C for 10 s, 72°C for 10 s; 2 cycles of 98°C for 10 s, 64°C for 10 s, 72°C for 10 s; 2 cycles of 98°C for 10 s, 61°C for 10 s, 72°C for 10 s; 31 cycles of 98°C for 10 s, 58°C for 10 s, 72°C for 10 s. PCR products were diluted, and quantified using the PicoGreen double-stranded DNA assay (Invitrogen).

    Article Title: High-throughput mutagenesis using a two-fragment PCR approach
    Article Snippet: .. PCR set-up and high-throughput mutagenesis PCR conditions were first tested for a random set of 30 CB2 mutants using Phusion High-Fidelity PCR Master Mix with HF Buffer, Phusion High-Fidelity PCR Master Mix with GC Buffer (New England Biolabs, NEB) and KOD Hot Start Master Mix (Merck Millipore). .. Based on analysis of the PCR products on agarose gels (Fig. ), Phusion High-Fidelity DNA Polymerase with GC buffer was chosen for high-throughput mutagenesis, as in most instances, it gave the expected DNA bands with no or very little non-specific products.

    Mutagenesis:

    Article Title: High-throughput mutagenesis using a two-fragment PCR approach
    Article Snippet: .. PCR set-up and high-throughput mutagenesis PCR conditions were first tested for a random set of 30 CB2 mutants using Phusion High-Fidelity PCR Master Mix with HF Buffer, Phusion High-Fidelity PCR Master Mix with GC Buffer (New England Biolabs, NEB) and KOD Hot Start Master Mix (Merck Millipore). .. Based on analysis of the PCR products on agarose gels (Fig. ), Phusion High-Fidelity DNA Polymerase with GC buffer was chosen for high-throughput mutagenesis, as in most instances, it gave the expected DNA bands with no or very little non-specific products.

    Size-exclusion Chromatography:

    Article Title: The Tsetse Fly Displays an Attenuated Immune Response to Its Secondary Symbiont, Sodalis glossinidius
    Article Snippet: .. RNAi-Mediated Imd-Pathway Suppression To construct the relish dsRNA, the complete relish coding sequence (2,599 bp) was first PCR amplified using the Phusion High-Fidelity PCR master mix (New England BioLabs) in a total volume of 50 μl and 0.5 μM of each primer ( ) with following cycling conditions: 30 sec./98°C, 35 cycles 10 sec./98°C, 30 sec./65°C, 30 sec./72°C, and 10 min/72°C. .. For this, cDNA was generated from total RNA isolated from 10-day old wild-type abdomens as described above.

    Construct:

    Article Title: The Tsetse Fly Displays an Attenuated Immune Response to Its Secondary Symbiont, Sodalis glossinidius
    Article Snippet: .. RNAi-Mediated Imd-Pathway Suppression To construct the relish dsRNA, the complete relish coding sequence (2,599 bp) was first PCR amplified using the Phusion High-Fidelity PCR master mix (New England BioLabs) in a total volume of 50 μl and 0.5 μM of each primer ( ) with following cycling conditions: 30 sec./98°C, 35 cycles 10 sec./98°C, 30 sec./65°C, 30 sec./72°C, and 10 min/72°C. .. For this, cDNA was generated from total RNA isolated from 10-day old wild-type abdomens as described above.

    Sequencing:

    Article Title: The Tsetse Fly Displays an Attenuated Immune Response to Its Secondary Symbiont, Sodalis glossinidius
    Article Snippet: .. RNAi-Mediated Imd-Pathway Suppression To construct the relish dsRNA, the complete relish coding sequence (2,599 bp) was first PCR amplified using the Phusion High-Fidelity PCR master mix (New England BioLabs) in a total volume of 50 μl and 0.5 μM of each primer ( ) with following cycling conditions: 30 sec./98°C, 35 cycles 10 sec./98°C, 30 sec./65°C, 30 sec./72°C, and 10 min/72°C. .. For this, cDNA was generated from total RNA isolated from 10-day old wild-type abdomens as described above.

    Article Title: Amplification of the MET receptor drives resistance to anti-EGFR therapies in colorectal cancer
    Article Snippet: .. The first amplification was performed in a 50 μl PCR reaction, 1× Phusion high-fidelity buffer, 1.5 U Hotstart Phusion polymerase (NEB, BioLabs), 0.5 μM of each primer with tag sequence, 0.2 mM of each deoxynucleoside triphosphate, and 0.5 mM MgCl2. .. Amplification was carried out using the following cycling conditions: 98°C for 45 s; 2 cycles of 98 °C for 10 s, 67°C for 10 s, 72°C for 10 s; 2 cycles of 98°C for 10 s, 64°C for 10 s, 72°C for 10 s; 2 cycles of 98°C for 10 s, 61°C for 10 s, 72°C for 10 s; 31 cycles of 98°C for 10 s, 58°C for 10 s, 72°C for 10 s. PCR products were diluted, and quantified using the PicoGreen double-stranded DNA assay (Invitrogen).

    Concentration Assay:

    Article Title: High-throughput sequence analysis reveals variation in the relative abundance of components of the bacterial and fungal microbiota in the rhizosphere of Ginkgo biloba
    Article Snippet: .. Each 30 µl PCR reaction mixture contained 5∼10 ng DNA template, 15 µl 2 × Master Mix (Phusion® High-Fidelity PCR Master Mix with GC Buffer, New England Biolabs, USA), with each primer in the reaction mixture being supplied at a concentration of 3 µM. ..

    High Throughput Screening Assay:

    Article Title: High-throughput mutagenesis using a two-fragment PCR approach
    Article Snippet: .. PCR set-up and high-throughput mutagenesis PCR conditions were first tested for a random set of 30 CB2 mutants using Phusion High-Fidelity PCR Master Mix with HF Buffer, Phusion High-Fidelity PCR Master Mix with GC Buffer (New England Biolabs, NEB) and KOD Hot Start Master Mix (Merck Millipore). .. Based on analysis of the PCR products on agarose gels (Fig. ), Phusion High-Fidelity DNA Polymerase with GC buffer was chosen for high-throughput mutagenesis, as in most instances, it gave the expected DNA bands with no or very little non-specific products.

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  • 99
    New England Biolabs phusion high fidelity pcr master mix
    Agarose gel electrophoresis analysis of <t>PCR</t> fragments multiplied by <t>Phusion</t> High-Fidelity PCR Master Mix with HF Buffer and Phusion High-Fidelity PCR Master Mix with GC Buffer. Analysed PCRs are labelled as the mutated residue and the letter indicating whether the fragment is multiplied by mutation-specific forward (F) or reverse (R) primer. Expected PCR fragments are between 3000 and 4000 bp long. While using the PCR master mix with GC buffer gave expected fragments in all shown cases, using the Phusion High-Fidelity polymerase with HF buffer failed to multiply four fragments.
    Phusion High Fidelity Pcr Master Mix, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 739 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phusion high fidelity pcr master mix/product/New England Biolabs
    Average 99 stars, based on 739 article reviews
    Price from $9.99 to $1999.99
    phusion high fidelity pcr master mix - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    88
    New England Biolabs phusion flash master mix
    Preparation of paired-end sequencing libraries from Sordaria macrospora. ( a ) and sheared by sonication with a Branson 450 Sonifier with microtip (duty cycle 80%, output 1.2; five cycles of 10 s pulses, interrupted by 30 s rest on ice). Sheared DNA was concentrated (Qiagen PCR purification kit) and separated on a 1.2% agarose gel. Gel slices of ∼300 and ∼500 bp fragments were isolated and purified (Qiagen Gel extraction kit, see Subheading 3.4, step 5). ( b ) Paired-end libraries were constructed as described in the methods from the 300- and 500-bp fractions and amplified with 12 cycles of PCR, yielding barely visible bands. Note presence of adapters and PCR primers below the 100 bp marker. ( c ) Test for library complexity. One simple test to show that libraries contain predominantly genomic DNA and are not enriched for adapter dimers or other spurious overamplified bands is to digest the DyNAzyme-amplified libraries with Dpn II. This cleaves most of the adapter off the inserts. If genomic DNA has been cloned it will result in a smear (similar to genomic DNA digested for Southern blots). If only low complexity samples have been cloned in the library, sharp banding is observed instead of the smear and the library is unsuitable for further processing. ( d ) Libraries were amplified with 18 cycles of PCR with <t>Phusion</t> Flash High-Fidelity polymerase and purified (Qiagen PCR purification kit; see three 300-bp library samples). We typically pool three independent 25 μmL PCR reactions before purification and run 5 μL of 35 μL to show that only expected bands are obtained. Note the absence of the adapters or PCR primers and primer dimers (compare to ( b )).
    Phusion Flash Master Mix, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phusion flash master mix/product/New England Biolabs
    Average 88 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    phusion flash master mix - by Bioz Stars, 2020-08
    88/100 stars
      Buy from Supplier

    Image Search Results


    Agarose gel electrophoresis analysis of PCR fragments multiplied by Phusion High-Fidelity PCR Master Mix with HF Buffer and Phusion High-Fidelity PCR Master Mix with GC Buffer. Analysed PCRs are labelled as the mutated residue and the letter indicating whether the fragment is multiplied by mutation-specific forward (F) or reverse (R) primer. Expected PCR fragments are between 3000 and 4000 bp long. While using the PCR master mix with GC buffer gave expected fragments in all shown cases, using the Phusion High-Fidelity polymerase with HF buffer failed to multiply four fragments.

    Journal: Scientific Reports

    Article Title: High-throughput mutagenesis using a two-fragment PCR approach

    doi: 10.1038/s41598-017-07010-4

    Figure Lengend Snippet: Agarose gel electrophoresis analysis of PCR fragments multiplied by Phusion High-Fidelity PCR Master Mix with HF Buffer and Phusion High-Fidelity PCR Master Mix with GC Buffer. Analysed PCRs are labelled as the mutated residue and the letter indicating whether the fragment is multiplied by mutation-specific forward (F) or reverse (R) primer. Expected PCR fragments are between 3000 and 4000 bp long. While using the PCR master mix with GC buffer gave expected fragments in all shown cases, using the Phusion High-Fidelity polymerase with HF buffer failed to multiply four fragments.

    Article Snippet: PCR set-up and high-throughput mutagenesis PCR conditions were first tested for a random set of 30 CB2 mutants using Phusion High-Fidelity PCR Master Mix with HF Buffer, Phusion High-Fidelity PCR Master Mix with GC Buffer (New England Biolabs, NEB) and KOD Hot Start Master Mix (Merck Millipore).

    Techniques: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Mutagenesis

    'PER' genome-wide base composition bias curves . (a,b) Shown is the GC bias in Illumina reads from a 400-bp fragment library amplified using the standard PCR protocol (Phusion HF, short denaturation) on a fast-ramping thermocycler (red squares), Phusion HF with long denaturation and 2M betaine (black triangles), AccuPrime Taq HiFi with long denaturation and primer extension at 65°C (blue diamonds) or 60°C (purple diamonds). To calculate the observed to expected (unbiased) read coverage, the number of reads aligning to 50-bp windows at a given %GC was divided by the number of 50-bp windows that fall in this %GC category. This value was then normalized relative to the average value from 48% through 52% GC and plotted on a log 10 scale (a) or linear scale (b).

    Journal: Genome Biology

    Article Title: Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries

    doi: 10.1186/gb-2011-12-2-r18

    Figure Lengend Snippet: 'PER' genome-wide base composition bias curves . (a,b) Shown is the GC bias in Illumina reads from a 400-bp fragment library amplified using the standard PCR protocol (Phusion HF, short denaturation) on a fast-ramping thermocycler (red squares), Phusion HF with long denaturation and 2M betaine (black triangles), AccuPrime Taq HiFi with long denaturation and primer extension at 65°C (blue diamonds) or 60°C (purple diamonds). To calculate the observed to expected (unbiased) read coverage, the number of reads aligning to 50-bp windows at a given %GC was divided by the number of 50-bp windows that fall in this %GC category. This value was then normalized relative to the average value from 48% through 52% GC and plotted on a log 10 scale (a) or linear scale (b).

    Article Snippet: Standard reactions contained 1× Phusion High-Fidelity PCR master mix with HF buffer (NEB).

    Techniques: Genome Wide, Amplification, Polymerase Chain Reaction

    Effect of temperature ramp rates . The standard PCR protocol with Phusion HF DNA polymerase and short initial (30 s) and in-cycle (10 s) denaturation times was performed on three different thermocyclers at their respective default temperature ramp settings. Heating and cooling rates were 6°C/s and 4.5°C/s on thermocycler 1 (bright red line), 4°C/s and 3°C/s on thermocycler 2 (purple line) and 2.2°C/s and 2.2°C/s on thermocycler 3 (dark red line).

    Journal: Genome Biology

    Article Title: Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries

    doi: 10.1186/gb-2011-12-2-r18

    Figure Lengend Snippet: Effect of temperature ramp rates . The standard PCR protocol with Phusion HF DNA polymerase and short initial (30 s) and in-cycle (10 s) denaturation times was performed on three different thermocyclers at their respective default temperature ramp settings. Heating and cooling rates were 6°C/s and 4.5°C/s on thermocycler 1 (bright red line), 4°C/s and 3°C/s on thermocycler 2 (purple line) and 2.2°C/s and 2.2°C/s on thermocycler 3 (dark red line).

    Article Snippet: Standard reactions contained 1× Phusion High-Fidelity PCR master mix with HF buffer (NEB).

    Techniques: Polymerase Chain Reaction

    Optimizing the PCR conditions . (a) Neither extending the denaturation times (dark red squares) nor adding 2M betaine (black triangles) is sufficient to recover extremely GC-rich DNA fragments by PCR with Phusion HF. (b) Combining long denaturation and 2M betaine is effective for the high-GC fraction (black triangles) but the profile is not as even over the entire GC spectrum as after PCR with AccuPrime Taq HiFi (blue diamonds) using extended denaturation times and a lower temperature (65°C) for primer annealing and extension.

    Journal: Genome Biology

    Article Title: Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries

    doi: 10.1186/gb-2011-12-2-r18

    Figure Lengend Snippet: Optimizing the PCR conditions . (a) Neither extending the denaturation times (dark red squares) nor adding 2M betaine (black triangles) is sufficient to recover extremely GC-rich DNA fragments by PCR with Phusion HF. (b) Combining long denaturation and 2M betaine is effective for the high-GC fraction (black triangles) but the profile is not as even over the entire GC spectrum as after PCR with AccuPrime Taq HiFi (blue diamonds) using extended denaturation times and a lower temperature (65°C) for primer annealing and extension.

    Article Snippet: Standard reactions contained 1× Phusion High-Fidelity PCR master mix with HF buffer (NEB).

    Techniques: Polymerase Chain Reaction

    Preparation of paired-end sequencing libraries from Sordaria macrospora. ( a ) and sheared by sonication with a Branson 450 Sonifier with microtip (duty cycle 80%, output 1.2; five cycles of 10 s pulses, interrupted by 30 s rest on ice). Sheared DNA was concentrated (Qiagen PCR purification kit) and separated on a 1.2% agarose gel. Gel slices of ∼300 and ∼500 bp fragments were isolated and purified (Qiagen Gel extraction kit, see Subheading 3.4, step 5). ( b ) Paired-end libraries were constructed as described in the methods from the 300- and 500-bp fractions and amplified with 12 cycles of PCR, yielding barely visible bands. Note presence of adapters and PCR primers below the 100 bp marker. ( c ) Test for library complexity. One simple test to show that libraries contain predominantly genomic DNA and are not enriched for adapter dimers or other spurious overamplified bands is to digest the DyNAzyme-amplified libraries with Dpn II. This cleaves most of the adapter off the inserts. If genomic DNA has been cloned it will result in a smear (similar to genomic DNA digested for Southern blots). If only low complexity samples have been cloned in the library, sharp banding is observed instead of the smear and the library is unsuitable for further processing. ( d ) Libraries were amplified with 18 cycles of PCR with Phusion Flash High-Fidelity polymerase and purified (Qiagen PCR purification kit; see three 300-bp library samples). We typically pool three independent 25 μmL PCR reactions before purification and run 5 μL of 35 μL to show that only expected bands are obtained. Note the absence of the adapters or PCR primers and primer dimers (compare to ( b )).

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

    Article Title: Library Preparation and Data Analysis Packages for Rapid Genome Sequencing

    doi: 10.1007/978-1-62703-122-6_1

    Figure Lengend Snippet: Preparation of paired-end sequencing libraries from Sordaria macrospora. ( a ) and sheared by sonication with a Branson 450 Sonifier with microtip (duty cycle 80%, output 1.2; five cycles of 10 s pulses, interrupted by 30 s rest on ice). Sheared DNA was concentrated (Qiagen PCR purification kit) and separated on a 1.2% agarose gel. Gel slices of ∼300 and ∼500 bp fragments were isolated and purified (Qiagen Gel extraction kit, see Subheading 3.4, step 5). ( b ) Paired-end libraries were constructed as described in the methods from the 300- and 500-bp fractions and amplified with 12 cycles of PCR, yielding barely visible bands. Note presence of adapters and PCR primers below the 100 bp marker. ( c ) Test for library complexity. One simple test to show that libraries contain predominantly genomic DNA and are not enriched for adapter dimers or other spurious overamplified bands is to digest the DyNAzyme-amplified libraries with Dpn II. This cleaves most of the adapter off the inserts. If genomic DNA has been cloned it will result in a smear (similar to genomic DNA digested for Southern blots). If only low complexity samples have been cloned in the library, sharp banding is observed instead of the smear and the library is unsuitable for further processing. ( d ) Libraries were amplified with 18 cycles of PCR with Phusion Flash High-Fidelity polymerase and purified (Qiagen PCR purification kit; see three 300-bp library samples). We typically pool three independent 25 μmL PCR reactions before purification and run 5 μL of 35 μL to show that only expected bands are obtained. Note the absence of the adapters or PCR primers and primer dimers (compare to ( b )).

    Article Snippet:Phusion Flash master mix: 1 unit/μL Phusion Flash High-Fidelity polymerase, 50 mM TAPS-HCl (pH 9.3), 100 mM KCl, 3 mM MgCl2 , 2 mM β-mercaptoethanol, 400 μM of dATP, dCTP, dGTP, and dTTP (NEB).

    Techniques: Sequencing, Sonication, Polymerase Chain Reaction, Purification, Agarose Gel Electrophoresis, Isolation, Gel Extraction, Construct, Amplification, Marker, Clone Assay