Q5 high fidelity dna polymerase 2 master mix

PCR reactions were prepared using the Q5 High Fidelity DNA polymerase 2× Master Mix (New England Biolabs), 0.5 µM forward primer, 0.5 µM reverse primer, 20 ng of template plasmid and nuclease-free water up to 25 µl of total volume. The PCR program started with a 98 °C initial denaturation step for 10 minutes, followed by 98 °C for 15 seconds, 60 °C for 30 seconds, 72 °C for 1 minute for 20 cycles, followed by 72 °C for 10 minutes.

Reverse transcription products of GC in hEx45KI-mdx44 mice were PCR amplified with Q5 high-fidelity DNA polymerase 2× Master Mix (New England Biolabs) and forward (design of mouse Dmd exon 43) and reverse (design of mouse Dmd exon 46) primers and then purified with a QIAquick PCR Purification Kit according to the manufacturer’s instructions. Purified PCR products were amplified and purified with a BigDye terminatorv3.1 Cycle sequencing Kit according to the manufacturer’s instructions and sequenced with a 3500 Genetic analyzer by the Sanger method (Thermo Fisher Scientific).

According to the manufacturer’s instructions, DNA was extracted from the isolated strains using the GenElute bacterial genomic DNA kit (Sigma-Aldrich). The integrity of the extracted genomic DNA was visualized in a 1 % agarose gel, and the DNA concentration was measured by Qubit (Thermo Fisher Scientific).
The 16S ribosomal DNA was amplified by PCR using Q5 High-Fidelity DNA polymerase 2× master mix (NEB). The amplification program cycle was as described by Frank et al. [42 (link)] and Lau et al. [43 (link)], with AGTTTGATCCTGGCTCAG and TACCTTGTTACGACTT primers to amplify almost the entire 16S ribosomal DNA gene. The PCR reactions were made up of 25 µl as per the manufacturer’s instructions and the remainder of the PCR product was cleaned with a Monarch genomic DNA purification kit (NEB). The purified products were Sanger sequenced from both ends using the primers described above (Eurofins).
The Sanger sequences were trimmed for quality, and the forward and reverse reads were aligned using the MegaAlign Pro 17 software from DNASTAR using Clustal Omega as the alignment method to obtain a consensus sequence. The consensus sequence for each isolate was checked against the nucleotide collection (nr/nt) database at the National Center for Biotechnology Information (NCBI), using the blast (Basic Local Alignment Search Tool) network service (http://www.ncbi.nlm.nih.gov).