For PCR analysis, p16 microsatellite marker DS91747 with the sequence of forward 5’-ATTCAACG AGTGG GATGAAG-3’ and reverse 5’-TCCAGGTTGCTGCAAATGCC-3’ was selected. The PCR product size expected was 130–150 bp.
Ampli Taq red master mix containing 2 mM MgCl2, 0.2 mM of dNTPs and 1 U/μl of Taq DNA polymerase was prepared as reaction mixture. Primer concentration of 0.4 μM and 3 μl of DNA template was added to the mixture. To carry out amplification, a Veriti thermal cycler (Applied Biosystems, CA, USA) was used. After this, thermal cycling conditions were used for running the PCR which was 95°C for 5 min, followed by 40 cycles of 95°C for 30 s, 54°C for 1 min and 72°C for 1 min. Finally, 2% agarose gel was prepared and the 15 μl samples were loaded on to the gel which was further subjected to electrophoresis at 80 V for 1 h. The gel was subsequently stained with 0.5 μg/ml ethidium bromide and observed under ultraviolet gel documentation system (Major Science, Saratoga, CA, USA).[14 (link)]
The molecular weight of the band was confirmed by comparing the location with standard molecular weight marker ladder (100–1500 bp).[12 ]