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Hi
For a while I've been trying to amplify different regions on human chromosome 21 q arm. I'm using long primers (60-90 bp) with high theoretical Tm between 71 and 72 deg C.
I've tested several different annealing temperatures in the range from 65-69 deg C. With one primer pair I get a moderately strong PCR product at 69 deg C.The last attempt I made was a two stage PCR with annealing temperature combinations
69 C-10 cycles/70 C-15 cycles,
69 C-10 cycles/71 C -15 cycles,
69 C-10 cycles/72 C -15 cycles,
69 C-10 cycles/74 C -15 cycles
and it didn't work.
I'm using HiFi polymerase by Kapa Biosystems with GC buffer. The initial denaturation is 98 deg for 5 min. Denaturarion in each cycle is 98 deg for 20 sec. The manufacturer suggests using two stage PCR when long primer pairs are used.
Anyone has any advice regarding the amplification?
Thanks
