long primers PCR
Posted 10 January 2013 - 12:18 PM
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?
Posted 10 January 2013 - 05:46 PM
Posted 11 January 2013 - 01:30 AM
Posted 11 January 2013 - 02:19 AM
Except for the 12 bases at the 5 end side where I have my restriction site, the rest of the primers anneal to the target sequence.
I'm trying one more round with the Kapa HiFi strictly following their technical datasheet info. For the long primers which have the Ta above 68 deg they recommend two stage PCR setup but in my case it doesn't work.
If this last attempt doesn't work I'll switch to Pwo and see what happens.
Posted 15 January 2013 - 10:00 AM
But from the fact you only got PCR product with the higher temperatures I assume maybe the main problem is secondary structures on your (very long) primers. But since you already use GC buffer with probably addition of DMSO or some other secondary structures-relaxing reagent, it's probably useless to try add DMSO..
I never trust anything that can't be doubted.