i am amplifying a gene which is GC rich (5' highly GC rich ~76% and 3' ~66%). I have non-specific amplification but no desired band till now. I have done/used
1. gradient PCR
2. DMSO etc.
but no desired band (~2Kb).
PCR ingradients: DreamTaq GMM, 0.4 uM each primer, 200ng gDNA.
Profile: initaion denat. 95oC-5mint, 95oC-1 mint, 55-67oC for 1mint, 72oC for 2 mint.
FP Tm = 63.75
RP Tm = 57.88
Thanks.
Hello,
I work with high-GC (70-80%) streptomycete bacteria and regularly amplify their DNA.
Several points that may help:
1) Polymerase buffer - we tend to use either Phusion or Q5 polymerases with the High-GC optimised buffer (included when you purchase.
2) PCR protocol - gradient PCR amplifications can work well, but I would also try performing a two-step PCR (72 oC annealing temperature - same as the extension). The Tm of your primers might be a little low for this, but worth a shot.
3) DMSO is generally good to include (3-5%). I would also try adding 1M betaine ( or ethylene glycol to your reactions. Betaine destabilises GC binding; this has really helped my in several instances get a clean band of the correct size.
Good luck!
I am with Rory on this one...amplifying in the presence of 1M final concentration betaine ( very cheap from Sigma any grade works) and keeping the primer annealing temperature as high as possible has often worked for me. If using betaine I would try without dmso first and then using dmso. If this fails (and always a good practice) check for SNPs lying under one primer and consider nested primers for a second round amplification on the assumption that it has worked but not very well
Hello
Rory and ninety said quite correctly, Just note that the betaine and dmso do not use together at first
Good luck!
Other than DMSO, GC buffer, and relatively high annealing temperature, you can also optimize in terms of template: use genome DNA or heat the bacteria at 95C for 10min before used.
@Dear Menghao Yu,
thanks. i heat the sample at 95C for 10 min but band amplification not occure or a little. perhaps degradation.
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