Jumpstart red taq polymerase

We extracted genomic DNA from murine and human GC cell lines by the phenol-chloroform method, and then carried out bisulfite modification and the MSP procedure. Bisulfite treatment of DNA was performed with EZ DNA Methylation-Gold (ZYMO RESEARCH; #D5006), and then methylation-specific PCR (MSP) was conducted. Briefly, the PCR reaction was performed for 35 cycles in a 25 μl mixture comprising bisulfite-modified DNA, 2.5μl of 10x PCR buffer, 1.25 μl of 25mM dNTPs, 25 pmol/l of each primer and 1 U of JumpStart RedTaq polymerase (Sigma; #D0563). The conditions for PCR were as follows: 95°C for 5 minutes; 35 cycles of 95°C for 1minutes, 53°C for 2minutes, and 72°C for 1.5minutes; and a final extension at 72°C for 5 minutes. As for mouse GC tissues, DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissues. Bisulfite DNA was amplified with flanking PCR primers and then nested MSP was conducted [25 (link), 26 (link)]. The primer sequences and PCR product sizes are shown in S2 Table.

ATF5 whole-body KO mice were generated by crossing ATF5^tm1(KOMP) (Velocigene Project 11,612) heterozygotes in a C57BL6/N background, generously provided by Dr. Stavros Lomvardas from Columbia University, with FVB WT females. Animals were housed in a 12:12-h light–dark cycle and given food and water ad libitum. To genotype progeny, ear clippings were obtained from each animal to make crude DNA extracts. They were subsequently mixed with JumpStart REDTaq polymerase (P0982, Sigma), forward and reverse primers (50 $\mu$ M) (Table S1) for the WT and altered ATF5 gene, and subjected to amplification by PCR. Reaction products were run on a 1% agarose gel and visualized with the use of ethidium bromide. Experimental animals were used at 5.5–7 months of age and separated into either Control (CON) or Exercise (EX) groups (n = 9–13/group). For the PGC-1 $\alpha$ KO experiments, TA muscles were extracted from WT and whole-body PGC-1 $\alpha$ KO mice at 4–5 months of age [55 (link)], snap-frozen in liquid nitrogen and stored at −80 $°$ C for later analysis by qPCR (n = 3–5/group).