I am working on vitamin D receptor (VDR) gene, and my primers are:
Taq I: 5’-CCTTCTTCTCTATCCCCGTG-3’ (Forward) and 5’-GCAGGTCGGCTAGCTTCT-3’ (Reverse)
Fok I: 5’-ACTCTGGCTCTGACCGTG-3’ (Forward) and 5’-TCATAGCATTGAAGTGAAAGC-3’
I want to amplify the gene by PCR; I have tried many protocols and no band appears even though when I run the samples before the PCR I get clear bands
I am using Promega master mix 12.5 ul + 1ul reverse primer + 1ul forward primer + 10 ul DNA sample + 0.5 ul nuclease free waterà 25ul finishing solution
Initial denaturation at 94° C for 5 min, 35 cycles at 94° C for 30 s, 58° C for 30 s and 72° C for 60 s and one final cycle of extension at 72° C for 7 min…
I have changed Initial denaturation to 95° C once; also I have increased the annealing temperature to 59° C.
I have increased the primers in place of the nuclease free water and I have increased the DNA sample
What can I do to perfect my protocol?