phusion  (New England Biolabs)

Journal: PLoS ONE

Article Title: Variations of five eIF4E genes across cassava accessions exhibiting tolerant and susceptible responses to cassava brown streak disease

doi: 10.1371/journal.pone.0181998

Figure Lengend Snippet: RT-PCR amplification of five cassava eIF4E ORFs with gene-specific primers. Total RNA was extracted from TMS60444 cassava line. The first-strand cDNA was synthesized using SuperScript ™ III Reverse Transcriptase (Invitrogen) and PCR was performed using Phusion High-Fidelity DNA polymerase (New England Biolabs) and primers indicated in Table 1 . Lane 1: DNA marker; Lane 2: 016601 CDS amplified with primers 016601F and 016601R; Lane 3: 016620 CDS amplified with primers 016620F and 016620R; Lane 4: 015501 CDS amplified with 015501F and 015501R, Lane 5: 013223 CDS amplified with primers 013223lF and 013223R, Lane 6, 013223 CDS amplified with primers 013223sF and 013223R; Lane 7: 013732 CDS amplified with 013732lF and 013732R; Lane 8: 013732 CDS amplified with 013732sF and 013732R; and Lane 9: negative water control.

Article Snippet: PCR was performed in a 20 μl reaction volume containing 10 unit Phusion DNA polymerase (NEB, Ipswich, MA), 1 μl of 1:5 diluted cDNA template, 1X Phusion PCR buffer, 5 μM each of upstream and downstream primers, and 250 nM dNTP with the following cycling condition: 98°C for 1 minute; 35 cycles of 98°C for 15 seconds, 56°C for 15 seconds, and 72°C for 45 seconds; and finally 72°C for 5 minutes.

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Synthesized, Polymerase Chain Reaction, Marker

Journal: PLoS ONE

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

doi: 10.1371/journal.pone.0156478

Figure Lengend 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.

Article Snippet: To address high GC content various additives were included in the Phusion HF PCR reactions as follows: 7-deaza-dGTP (NEB) at a 40:60 ratio with normal dGTP, as well as 50:50 and 60:40 ratios keeping the final concentration of dNTPs constant; DMSO (Sigma) at a final concentration of 2.5%, 5%, and 10%; betaine (Sigma) at a final concentration of 1M, 2M and 4M.

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

Journal: Scientific Reports

Article Title: PrimerMapper: high throughput primer design and graphical assembly for PCR and SNP detection

doi: 10.1038/srep20631

Figure Lengend Snippet: Validation and testing of PrimerMapper. ( a ) Primers were designed using PrimerMapper to span the C. elegans transcript, ZK5204a, and PCR products from a series of PCR reactions using these primers were examined by gel electrophoresis. Primer sequences are displayed in Table 1 . Robust bands of the correct size were obtained from each PCR. The first and last lanes are a GenRuler DNA ladder mix (Thermo Fisher Scientific, Waltham, MA). Primer parings starting from the left second lane were: F2 + R1, F2 + R2, F2 + R3, F2 + R4, F2 + R5, F2 + R6, F3 + R1, F3 + R2, F3 + R3, F3 + R4, F1 + R2, F1 + R3, F1 + R4, F1 + R5, F1 + R6, F1 + R8, F3 + R9, F5 + R6. ( b ) Files containing different numbers of sequences (2, 10, 20, 50, 100, 150, 200, 1,000) were provided as input to PrimerMapper and ran using default settings to generate primer text files for each sequence and graphic file generation input text files (i.e. Step 1 of execution – see Fig. 2 ). The resulting data is plotted with time (seconds) on the x-axis and the number of sequences on the y-axis. Fitting the relationship between sequence number and run-time with a quadratic equation yields an R 2 value of 0.9995. ( c–f) Comparison of the melting temperatures obtained for PrimerMapper from 100 randomly generated primers (18–30bps in size) with that of the NEB calculator for NEB Taq DNA Polymerase (c) , NEB Phusion ® Polymerase ( d ), NEB Q5 ® Hi-Fi Polymerase (e) , and with Primer3 16 ( f ).

Article Snippet: In each case, there were robust correlations observed between the melting temperatures calculated by PrimerMapper and each algorithm, with the highest correlation observed for PrimerMapper to that of the NEB calculator for NEB Phusion® polymerase ( ; r 2 = 0.99).

Techniques: Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Sequencing, Generated