phusion gc buffer  (New England Biolabs)

Journal: Nucleic Acids Research

Article Title: Restriction enzyme-free mutagenesis via the light regulation of DNA polymerization

doi: 10.1093/nar/gkp150

Figure Lengend Snippet: Effects of a caged thymidine nucleobase on the PCR. ( A ) PCR product using Phusion DNA Polymerase. Polymerization is halted in the presence of a caging group, which is confirmed using the truncated primer P3 that affords the same length product. ( B ) PCR product using Taq DNA polymerase. Polymerization is halted in the presence of a caging group, which is confirmed using the truncated primer P3 that affords the same length product.

Article Snippet: 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).

Techniques: Polymerase Chain Reaction

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: 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).

Techniques: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Mutagenesis