Pcr mastercycler

Total RNA was isolated using a TRIzol kit (Invitrogen, Carlsbad, CA). Complementary DNAs (cDNAs) were synthesized using 500 ng of total RNA with Oligo-(dT) 18 primer at 42°C for 30 min and terminated by heating at 85°C for 5 min. Primers were synthesized by Sangon (Shanghai China, Table 1). PCR amplification was performed at 94°C for 5 min, 54°C for 30 seconds, and 72°C for 90 seconds with 35 cycles with a PCR Mastercycler (Eppendorf, Hamburg, Germany). The products were analyzed by Genesnap software on a 1.5% agarose gel containing 10 mg/mL ethidium bromide. Bax, bcl-2, and caspase-3 mRNA levels were quantified in relation to β-actin mRNA level.

PCR was carried out to validate the identified high-quality nsSNPs. Genomic DNA for the sequencing reactions was extracted using the Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA). The amplification of the selected genes was performed using a standard PCR protocol. Each 25 μl PCR reaction contained 150 μM (each) deoxynucleoside triphosphates, 1× PCR colourless buffer, 1.2 mM MgCl₂, 0.2 μM of primer and 0.5 U of Go Taq Flexi DNA Polymerase (Promega, Madison, WI, USA). The PCR was performed under the following conditions: initial denaturation at 95°C for 30 s, 30 cycles of denaturation at 95°C for 30 s, 30 s at the respective annealing temperature (Additional file 10) and an extension step at 72°C for 40 s; a final extension was performed at 72°C at 1 min. The reactions were carried out using a PCR Master Cycler (Eppendorf AG, Hamburg, Germany). The primer sets that were used for the target genes are shown (Additional file 10).