phusion  (New England Biolabs)


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  • 99
    Name:
    Phusion GC Buffer Pack
    Description:
    Phusion GC Buffer Pack 6 0 ml
    Catalog Number:
    B0519S
    Price:
    24
    Category:
    Buffers
    Size:
    6 0 ml
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    Structured Review

    New England Biolabs phusion
    Phusion GC Buffer Pack
    Phusion GC Buffer Pack 6 0 ml
    https://www.bioz.com/result/phusion/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phusion - by Bioz Stars, 2021-05
    99/100 stars

    Images

    1) Product Images from "RF-Cloning.org: an online tool for the design of restriction-free cloning projects"

    Article Title: RF-Cloning.org: an online tool for the design of restriction-free cloning projects

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks396

    RF-Cloning.org output page. (1) A unique 32 byte hash code is generated for all new projects, and is present in the URL for bookmarking purposes. (2) The hybrid primers are color coded, blue for sequence complementary to the plasmid, and green for the insert. The length of the primers can be adjusted by clicking on the arrow buttons if the user wishes to alter the annealing temperature. (3) If the insert site needs to be adjusted, the user can use the provided arrow buttons. (4) The secondary PCR conditions are optimized for iProof or Phusion as the polymerase, so the user should follow manufacturer’s instructions if using another high fidelity enzyme. ‘Insert’ refers to the mega-primer purified from the primary PCR reaction. (5) The entire sequence of the new plasmid is output, with insert in green and parental plasmid in blue. (6) The plasmid map can be drawn by specifying the positions of markers manually, or by auto-finding common features. Restriction enzyme cut sites can also be specified or automatically identified. If desired, the plasmid can be exported as a genbank file. (7) All projects are automatically saved, but making changes to the output page will activate the save button so those changes can be uploaded to the database. If the user has registered an account to access the plasmid management system, the save button will attach the project to their profile. (8) After the project has been completed and sent for sequencing, the sequencing results can be copied into a popup window for BLAST2 sequence alignment.
    Figure Legend Snippet: RF-Cloning.org output page. (1) A unique 32 byte hash code is generated for all new projects, and is present in the URL for bookmarking purposes. (2) The hybrid primers are color coded, blue for sequence complementary to the plasmid, and green for the insert. The length of the primers can be adjusted by clicking on the arrow buttons if the user wishes to alter the annealing temperature. (3) If the insert site needs to be adjusted, the user can use the provided arrow buttons. (4) The secondary PCR conditions are optimized for iProof or Phusion as the polymerase, so the user should follow manufacturer’s instructions if using another high fidelity enzyme. ‘Insert’ refers to the mega-primer purified from the primary PCR reaction. (5) The entire sequence of the new plasmid is output, with insert in green and parental plasmid in blue. (6) The plasmid map can be drawn by specifying the positions of markers manually, or by auto-finding common features. Restriction enzyme cut sites can also be specified or automatically identified. If desired, the plasmid can be exported as a genbank file. (7) All projects are automatically saved, but making changes to the output page will activate the save button so those changes can be uploaded to the database. If the user has registered an account to access the plasmid management system, the save button will attach the project to their profile. (8) After the project has been completed and sent for sequencing, the sequencing results can be copied into a popup window for BLAST2 sequence alignment.

    Techniques Used: Clone Assay, Polyacrylamide Gel Electrophoresis, Generated, Sequencing, Plasmid Preparation, Polymerase Chain Reaction, Purification

    Related Articles

    Polymerase Chain Reaction:

    Article Title: RF-Cloning.org: an online tool for the design of restriction-free cloning projects
    Article Snippet: RECOMMENDED RF-CLONING PROTOCOL By default, the starting hybrid primers will be at least 40 bp long with an annealing temperature of at least 55°C for the primary PCR (amplification of insert) and at least 60°C for the secondary PCR (extension around the plasmid). .. High-fidelity DNA polymerase should be used for all PCR reactions, and it has been our experience that iProof (BioRad, Hercules, CA, USA) and Phusion (New England Biolabs, Ipswich, MA, USA) produce consistent results. .. To generate the mega-primer, use a standard 50 μl PCR reaction (1× PCR buffer, 200 μM dNTP, 500 nM of each primer, 1 U polymerase, user defined amount of starting template) and cycle 30–35 times with the RF-Cloning.org recommended annealing temperature, followed by product purification (e.g. by gel extraction).

    Article Title: High-throughput mutagenesis using a two-fragment PCR approach
    Article Snippet: When using KOD Hot Start Master Mix, the following thermocycling setup was used: Initial denaturation at 95 °C for 2 min, followed by 20 step-down thermal cycles, each comprising denaturation at 95 °C for 20 s, annealing from 65 °C down to 55.5 °C for 10 s (0.5 °C decrement per cycle), and elongation at 70 °C for at the appropriate time (up to 135 s), then 5 thermal cycles with the constant annealing temperature (denaturation at 95 °C for 20 s, annealing at 62.5 °C for 10 s, elongation at 70 °C with the same time period as used in the step-down cycles), completed with a hold at 10 °C. .. For truncations of the AtETR1 and LeETR1 plasmids by the two-fragment approach, a slightly different PCR protocol was used: 20 μL PCR mixture prepared with nuclease-free water (Cell Signaling Technology) contained 1 ng DNA template, 500 nM each primer, 200 μM each dNTP (deoxynucleoside triphosphate; NEB), 0.4 U μL−1 Phusion High-Fidelity DNA Polymerase (NEB), and either 1× Phusion buffer HF (NEB) or 3% (v/v) DMSO (NEB) with 1× Phusion buffer GC (NEB). .. Also, PCR thermocycling described above was modified in case of the ETR1 plasmids to have: (1) 11 step-down cycles with annealing starting from 65 °C down to 60 °C (i.e. decreasing by 0.5 °C in each subsequent cycle); (2) elongation time of at least 30 s per kbp of the target product (up to 200 s for the longest PCR products); and (3) final elongation time of 5 min.

    Article Title: Restriction enzyme-free mutagenesis via the light regulation of DNA polymerization
    Article Snippet: It is important to note that the selection of primers is key, and for some sequences, longer hybridization arms are required (see GFP deletion in ), which in turn requires additional caging groups to prevent undesirable hybridization during the PCR (see ‘Results and Discussion’ section). .. The forward and reverse primers (5 μl each of a 10 μM solution), the pGFPuv template (1 μl of a 0.1 ng/μl solution), dNTPs (2 μl each of a 1 mM solution), Phusion GC Buffer (5 μl of a 10× stock, New England Biolabs), Phusion DNA Polymerase (1 μl, two units) and dH2 O (31 μl) were mixed and subjected to the following PCR program: 95°C (2 min), followed by 40 cycles of 95°C (30 s), 40°C (60 s) and 72°C (3.3 min), with a final extension at 72°C (2 min). .. An identical PCR reaction was then repeated, using 5 μl of the previous reaction as the template, followed by purification with a PCR cleanup kit (Promega).

    Article Title: Effects of wine-cap Stropharia cultivation on soil nutrients and bacterial communities in forestlands of northern China
    Article Snippet: .. PCR-based amplifications were performed using Phusion® High-Fidelity PCR Master Mix with GC Buffer and high-fidelity DNA polymerase (New England Biolabs, Ipswich, MA, USA) following an amplification programme of 1 cycle at 98 °C for 1 min, 30 cycles composed of three steps for each cycle (98 °C for 10 s, 50 °C for 30 s, and 72 °C for 30 s), and a final elongation step of 72 °C for 5 min. . ..

    Article Title: In situ 10-cell RNA sequencing in tissue and tumor biopsy samples
    Article Snippet: Poly(A) PCR Poly(A) PCR was carried out with several modifications to the earlier procedure , . .. To each tailed sample, 90 µl of poly(A) PCR buffer was added to a final concentration of 1x ThermoPol buffer (New England Biolabs), 2.5 mM MgSO4 , 1 mM dNTPs (Roche), 100 µg/ml BSA (Roche), 3.75 U Taq polymerase (NEB) and 1.5 U Phusion (NEB) and 2.5 µg AL1 primer (ATTGGATCCAGGCCGCTCTGGACAAAATATGAATTCTTTTTTTTTTTTTTTTTTTTTTTT). .. Each reaction was split into three thin-walled 0.2 ml PCR tubes and amplified according to the following thermal cycling scheme: four cycles of 1 min at 94 °C (denaturation), 2 min at 32 °C (annealing) and 2 min plus 10 sec per cycle at 72 °C (extension); 21 cycles of 1 min at 94 °C (denaturation), 2 min at 42 °C (annealing) and 2 min 40 sec plus 10 sec per cycle at 72 °C (extension).

    Hybridization:

    Article Title: Solid-phase cloning for high-throughput assembly of single and multiple DNA parts
    Article Snippet: .. Extension An extension mix containing 17.25 μl water, 2.5 μl Phusion buffer (10×), 2.5 μl dNTPs (2 mM) and 0.25 μl Phusion (2 U/ μl New England Biolabs, Ipswich, MA, USA) was pre-heated at 65°C before used to resuspend the beads from the hybridization. .. Phusion HF was used as preferred buffer for extension unless the insert was PCR amplified using a different buffer; in those instances the matching buffer (e.g. Phusion GC buffer) was used also during the extension.

    Amplification:

    Article Title: Effects of wine-cap Stropharia cultivation on soil nutrients and bacterial communities in forestlands of northern China
    Article Snippet: .. PCR-based amplifications were performed using Phusion® High-Fidelity PCR Master Mix with GC Buffer and high-fidelity DNA polymerase (New England Biolabs, Ipswich, MA, USA) following an amplification programme of 1 cycle at 98 °C for 1 min, 30 cycles composed of three steps for each cycle (98 °C for 10 s, 50 °C for 30 s, and 72 °C for 30 s), and a final elongation step of 72 °C for 5 min. . ..

    Concentration Assay:

    Article Title: In situ 10-cell RNA sequencing in tissue and tumor biopsy samples
    Article Snippet: Poly(A) PCR Poly(A) PCR was carried out with several modifications to the earlier procedure , . .. To each tailed sample, 90 µl of poly(A) PCR buffer was added to a final concentration of 1x ThermoPol buffer (New England Biolabs), 2.5 mM MgSO4 , 1 mM dNTPs (Roche), 100 µg/ml BSA (Roche), 3.75 U Taq polymerase (NEB) and 1.5 U Phusion (NEB) and 2.5 µg AL1 primer (ATTGGATCCAGGCCGCTCTGGACAAAATATGAATTCTTTTTTTTTTTTTTTTTTTTTTTT). .. Each reaction was split into three thin-walled 0.2 ml PCR tubes and amplified according to the following thermal cycling scheme: four cycles of 1 min at 94 °C (denaturation), 2 min at 32 °C (annealing) and 2 min plus 10 sec per cycle at 72 °C (extension); 21 cycles of 1 min at 94 °C (denaturation), 2 min at 42 °C (annealing) and 2 min 40 sec plus 10 sec per cycle at 72 °C (extension).

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    New England Biolabs phusion buffer
    Results from head-to-tail SPC. ( A ) Comparison of compatibility of thermostable polymerases with the head-to-tail method. The polymerases with best proof-reading capabilities, <t>Phusion</t> and Deep Vent, all failed to generate transformants with the standard head-to-tail protocol. ( B ) Activity was retained for protocols using Phusion after supplementation of the washing buffer with SDS. ( C ) Colony screens of inserted region representing a selection of assemblies of various lengths and number of inserts assembled by head-to-tail SPC. Final construct sizes spanned 2.9 to 7.2 kbps.
    Phusion Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phusion buffer/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    99
    New England Biolabs phusion high fidelity dna polymerase
    Cloned pre-mir-122 stem-loop region sequences from HepG2 <t>DNA</t> show two different haplotypes. (A) Cloned DNA sequences obtained after amplification with Taq polymerase. Two haplotypes (differently shaded) were observed for HepG2, consistent with the presence of two alleles across this region. However, among the eight HepG2 and Huh-7 clones, six sequence differences to the reference genome assembly were detected (*), so cloning was repeated using a proofreading DNA polymerase. (B) Cloned DNA sequences obtained after amplification with <t>Phusion</t> high fidelity DNA polymerase. Essentially the same two haplotypes of HepG2 were seen, but three novel single nucleotide substitution variants were detected and in a fourth clone, the rs9966765 allele did not correspond to the background haplotype observed. The reported error rate of Phusion High-Fidelity DNA Polymerase (GC Buffer) is 9.5 x 10 -7 errors / base pair / PCR cycle (New England Biolabs). SNPs rs9966765 and rs1135519 are located upstream of the pre-mir-122 stem-loop region; their respective alleles are shown. The genomic positions on chromosome 18 (GRCh37/hg19 (Feb. 2009) human genome assembly) of non-SNP sequence variants and the alleles observed are shown; (T) n refers to the length (base pairs) of the polymorphic poly(T) tract. *, position showing a sequence variant not corresponding to the predominant haplotypes observed.
    Phusion High Fidelity Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phusion high fidelity dna polymerase/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Results from head-to-tail SPC. ( A ) Comparison of compatibility of thermostable polymerases with the head-to-tail method. The polymerases with best proof-reading capabilities, Phusion and Deep Vent, all failed to generate transformants with the standard head-to-tail protocol. ( B ) Activity was retained for protocols using Phusion after supplementation of the washing buffer with SDS. ( C ) Colony screens of inserted region representing a selection of assemblies of various lengths and number of inserts assembled by head-to-tail SPC. Final construct sizes spanned 2.9 to 7.2 kbps.

    Journal: Nucleic Acids Research

    Article Title: Solid-phase cloning for high-throughput assembly of single and multiple DNA parts

    doi: 10.1093/nar/gkv036

    Figure Lengend Snippet: Results from head-to-tail SPC. ( A ) Comparison of compatibility of thermostable polymerases with the head-to-tail method. The polymerases with best proof-reading capabilities, Phusion and Deep Vent, all failed to generate transformants with the standard head-to-tail protocol. ( B ) Activity was retained for protocols using Phusion after supplementation of the washing buffer with SDS. ( C ) Colony screens of inserted region representing a selection of assemblies of various lengths and number of inserts assembled by head-to-tail SPC. Final construct sizes spanned 2.9 to 7.2 kbps.

    Article Snippet: Extension An extension mix containing 17.25 μl water, 2.5 μl Phusion buffer (10×), 2.5 μl dNTPs (2 mM) and 0.25 μl Phusion (2 U/ μl New England Biolabs, Ipswich, MA, USA) was pre-heated at 65°C before used to resuspend the beads from the hybridization.

    Techniques: Activity Assay, Selection, Construct

    ssDNA production by aPCR. ( a ) aPCR reactions were assembled with a 50-molar excess of a forward primer for the amplification of a 1,000 nt ssDNA fragment using the M13mp18 ssDNA plasmid as template, and with 10 different polymerases that were tested for highest yield of ssDNA production (upper band: expected dsDNA size is 1,000 bp; lower band: expected ssDNA size is 1,000 nt) as judged by agarose gel electrophoresis (right panel). QuantaBio AccuStart HiFi, polymerase (lane 2, boxed) produced the highest amount without overlapping dsDNA contaminants. 1. Accustart; 2. Accustart HiFi; 3. Accustart II; 4. AccuPrime; 5. GoTaq; 6. DreamTaq; 7. Phusion; 8. Platinum SuperFi; 9. Q5; 10. Tth polymerase. ( b ) Biochemical validation of ssDNA production by incubating 1,000 nt aPCR reaction products with the ssDNA-specific ExoI or S1 nucleases or dsDNA-specific restriction enzymes Eco RI and Nae I (left panel). Agarose gel electrophoresis of the digestion products as labeled by lane (right panel). M: Marker, C: aPCR product control, ExoI: exonuclease I, S1: S1 nuclease, Enz: Eco RI + Nae I. ( c ) NEB LongAmp was used to generate ssDNA up to 15,000 nt long using lambda phage dsDNA as template. Purification of the 10 kb fragment shows a single band of higher molecular weight than the M13mp18 ssDNA (7,249 nt). ( d ) The primer design algorithm aPrime was used to select primers for product sizes between 500 and 3,000 nt using M13mp18 ssDNA as template and the Quantabio Accustart HiFi enzyme. SYBR Safe stained agarose gels illuminated under blue light show dsDNA as yellow bands, while ssDNA show as orange bands.

    Journal: Scientific Reports

    Article Title: In vitro synthesis of gene-length single-stranded DNA

    doi: 10.1038/s41598-018-24677-5

    Figure Lengend Snippet: ssDNA production by aPCR. ( a ) aPCR reactions were assembled with a 50-molar excess of a forward primer for the amplification of a 1,000 nt ssDNA fragment using the M13mp18 ssDNA plasmid as template, and with 10 different polymerases that were tested for highest yield of ssDNA production (upper band: expected dsDNA size is 1,000 bp; lower band: expected ssDNA size is 1,000 nt) as judged by agarose gel electrophoresis (right panel). QuantaBio AccuStart HiFi, polymerase (lane 2, boxed) produced the highest amount without overlapping dsDNA contaminants. 1. Accustart; 2. Accustart HiFi; 3. Accustart II; 4. AccuPrime; 5. GoTaq; 6. DreamTaq; 7. Phusion; 8. Platinum SuperFi; 9. Q5; 10. Tth polymerase. ( b ) Biochemical validation of ssDNA production by incubating 1,000 nt aPCR reaction products with the ssDNA-specific ExoI or S1 nucleases or dsDNA-specific restriction enzymes Eco RI and Nae I (left panel). Agarose gel electrophoresis of the digestion products as labeled by lane (right panel). M: Marker, C: aPCR product control, ExoI: exonuclease I, S1: S1 nuclease, Enz: Eco RI + Nae I. ( c ) NEB LongAmp was used to generate ssDNA up to 15,000 nt long using lambda phage dsDNA as template. Purification of the 10 kb fragment shows a single band of higher molecular weight than the M13mp18 ssDNA (7,249 nt). ( d ) The primer design algorithm aPrime was used to select primers for product sizes between 500 and 3,000 nt using M13mp18 ssDNA as template and the Quantabio Accustart HiFi enzyme. SYBR Safe stained agarose gels illuminated under blue light show dsDNA as yellow bands, while ssDNA show as orange bands.

    Article Snippet: The following enzymes were purchased from the respective commercial providers to test enzymatic production of ssDNA: AccuStart™, AccuStart™ II, and AccuStart™ HiFi from Quantabio; Q5® hot start HiFi, Phusion®, LongAmp®, Deep Vent®, and Deep Vent® (exo-) from New England BioLabs Inc. (NEB); AccuPrime™, Platinum™ SuperFi™, Tth and DreamTaq™ from ThermoFisher Scientific Inc.; GoTaq® from Promega (Promega corp.); and LA Taq ® from Takara Bio.

    Techniques: Amplification, Plasmid Preparation, Agarose Gel Electrophoresis, Produced, Labeling, Marker, Purification, Molecular Weight, Staining

    Cloned pre-mir-122 stem-loop region sequences from HepG2 DNA show two different haplotypes. (A) Cloned DNA sequences obtained after amplification with Taq polymerase. Two haplotypes (differently shaded) were observed for HepG2, consistent with the presence of two alleles across this region. However, among the eight HepG2 and Huh-7 clones, six sequence differences to the reference genome assembly were detected (*), so cloning was repeated using a proofreading DNA polymerase. (B) Cloned DNA sequences obtained after amplification with Phusion high fidelity DNA polymerase. Essentially the same two haplotypes of HepG2 were seen, but three novel single nucleotide substitution variants were detected and in a fourth clone, the rs9966765 allele did not correspond to the background haplotype observed. The reported error rate of Phusion High-Fidelity DNA Polymerase (GC Buffer) is 9.5 x 10 -7 errors / base pair / PCR cycle (New England Biolabs). SNPs rs9966765 and rs1135519 are located upstream of the pre-mir-122 stem-loop region; their respective alleles are shown. The genomic positions on chromosome 18 (GRCh37/hg19 (Feb. 2009) human genome assembly) of non-SNP sequence variants and the alleles observed are shown; (T) n refers to the length (base pairs) of the polymorphic poly(T) tract. *, position showing a sequence variant not corresponding to the predominant haplotypes observed.

    Journal: PLoS ONE

    Article Title: Demonstration of the Presence of the “Deleted” MIR122 Gene in HepG2 Cells

    doi: 10.1371/journal.pone.0122471

    Figure Lengend Snippet: Cloned pre-mir-122 stem-loop region sequences from HepG2 DNA show two different haplotypes. (A) Cloned DNA sequences obtained after amplification with Taq polymerase. Two haplotypes (differently shaded) were observed for HepG2, consistent with the presence of two alleles across this region. However, among the eight HepG2 and Huh-7 clones, six sequence differences to the reference genome assembly were detected (*), so cloning was repeated using a proofreading DNA polymerase. (B) Cloned DNA sequences obtained after amplification with Phusion high fidelity DNA polymerase. Essentially the same two haplotypes of HepG2 were seen, but three novel single nucleotide substitution variants were detected and in a fourth clone, the rs9966765 allele did not correspond to the background haplotype observed. The reported error rate of Phusion High-Fidelity DNA Polymerase (GC Buffer) is 9.5 x 10 -7 errors / base pair / PCR cycle (New England Biolabs). SNPs rs9966765 and rs1135519 are located upstream of the pre-mir-122 stem-loop region; their respective alleles are shown. The genomic positions on chromosome 18 (GRCh37/hg19 (Feb. 2009) human genome assembly) of non-SNP sequence variants and the alleles observed are shown; (T) n refers to the length (base pairs) of the polymorphic poly(T) tract. *, position showing a sequence variant not corresponding to the predominant haplotypes observed.

    Article Snippet: The observed error rate was ~14-fold higher than that reported for Phusion High-Fidelity DNA Polymerase (GC Buffer) (New England Biolabs; ) which, if this error rate is correct, suggests that these changes are unlikely to have arisen solely as a result of PCR errors.

    Techniques: Clone Assay, Amplification, Sequencing, Polymerase Chain Reaction, Variant Assay

    Olig2.5 inhibited archaeal family B DNA polymerases. An E. coli DH5α colony harbouring the 2.6 kb upstream sequence of Olig2 in pGL3-Basic vector was cultured and 1 μl of the culture was directly used as template for PCR. Olig2F and OligTSSR were used as primers to amplify the 2 kb fragment. Olig2.5 was added to see its inhibition to various DNAP. DNAP: DNA polymerase. LA: LA Taq DNAP. Q5: Q5 High-Fidelity DNAP. PS: PrimeSTAR HS DNAP. GXL: PrimeSTAR GXL DNAP. Phusion: Phusion High-Fidelity DNAP. Cobuddy: Cobuddy Super Fidelity DNAP. KOD: KOD DNAP.

    Journal: Scientific Reports

    Article Title: TT(N)mGCCTC inhibits archaeal family B DNA polymerases

    doi: 10.1038/s41598-018-20127-4

    Figure Lengend Snippet: Olig2.5 inhibited archaeal family B DNA polymerases. An E. coli DH5α colony harbouring the 2.6 kb upstream sequence of Olig2 in pGL3-Basic vector was cultured and 1 μl of the culture was directly used as template for PCR. Olig2F and OligTSSR were used as primers to amplify the 2 kb fragment. Olig2.5 was added to see its inhibition to various DNAP. DNAP: DNA polymerase. LA: LA Taq DNAP. Q5: Q5 High-Fidelity DNAP. PS: PrimeSTAR HS DNAP. GXL: PrimeSTAR GXL DNAP. Phusion: Phusion High-Fidelity DNAP. Cobuddy: Cobuddy Super Fidelity DNAP. KOD: KOD DNAP.

    Article Snippet: Phusion® High-Fidelity DNA Polymerase (Phusion) and Q5® High-Fidelity DNA Polymerase (Q5) were from New England Biolabs.

    Techniques: Sequencing, Plasmid Preparation, Cell Culture, Polymerase Chain Reaction, Inhibition