I am currently trying to standardise FMR1 gene mutation by PCR method. the region is highly repititive for CGG repeats and highly gc rich. I am using long primers 35nt length with high GC content > 85%, and tm 80 and 92. I am using the roche expand long template pcr kit with buffer 2 and high fidelity polymerase with hotstart method . I tried the conditions as 98X10min, 98X45 sec,64X1 min,68x2min and 68x 7 sec. Itried betaine in the concentration of 1.7-2.2 molar and q soltion , DMSO 10 %, but did not get any ampification . Then i tried various mgcl2 with 2.5-4 millimol. still no band.
I carrried the reaction with qiagen hot start , still no amplification. IF anyone worked with it lease help me