Ex taq dna polymerase hot start version

For genotyping of wild-type and Shmt2-knockout mice, DNA samples from the tails of adult mice or embryos were prepared using the Maxwell 16 system (Promega, Madison, WI, USA). PCR was performed using a TaKaRa Ex Taq DNA polymerase Hot Start version (Takara Bio, Shiga, Japan) and the following primer set: 5′-gag ttg acc aaa act gcc ct-3′, and 5′-tca agc ccc ata aac tgg tc-3′. The amplified samples were digested with XcmI to identify a mutation in Shmt2^{14 (link)}.

Genomic DNA was isolated using QIAamp DNA Mini Kit (Qiagen) according to the manufacturer’s instructions. Bisulfite conversion of DNA (500 ng/reaction) was performed using EZ DNA Methylation Kit (Zymo Research, Irvine, CA, USA). Methylated and unmethylated DNAs were amplified in separate reactions with primers designed using the MethPrimer program, and primer sequences used for this analysis are listed in Supplementary Table S1. Methylation-specific PCR reactions were performed using the EX TaqDNA polymerase Hot-Start version (Takara, Shiga, Japan) according to the manufacturer’s instructions.

Four loci within the ‘Ca. L. asiaticus’ genome containing TACAGAA, CAGT, AGACACA, and TTTG motifs [20 (link), 21 (link), 27 (link)] were examined in this study. These motifs had been previously designated as 001, 002, 005, and 077, respectively, [21 (link)]. The fragments containing these loci were amplified by PCR using the primer sets listed in Table 1, as previously reported [21 (link)]. Additionally, another forward primer was designed in order to be easy to count the motif of VNTR ‘001’ by using a program available on the Primer3 website (http://frodo.wi.mit.edu/primer3/) (Table 1).
PCR was performed using a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA) in a 20-μL reaction mixture containing 1 μL of DNA template, 0.1 μM of each primer, 200 μM dNTP mixture, 1× PCR buffer, and 2.5 units of Ex Taq DNA polymerase Hot Start Version (TaKaRa, Shiga, Japan). The thermal cycling conditions were as follows: initial denaturation at 92°C for 2 min; 35 cycles of denaturing at 92°C for 30 s, annealing at 54°C for 30 s, and extension at 72°C for 1 min. Amplified PCR products were separated by electrophoresis in a 1.5% (wt/vol) agarose gel in Tris-boric acid EDTA buffer. Before direct sequencing, the PCR products were extracted from the gel slices using a QIAquick Gel Extraction Kit (Qiagen) according to the manufacturer’s instructions.