Sometimes the whole gene region contains high GC content[~80%],it is difficult for Real Time PCR to analyze the simples;I have tried to design several primers to optimizate the results,but they all work not well. Can somebody give me some advise such as adding some additional agent or conduct other special methods?
P.S:I use the ABI7500-Fast Real Time PCR Instrument,and the SYBR® Premix Ex Taq™ GC [TAKARA] agent for reaction.
Dear Mao, You can include 0.25-0.4% high quality DMSO in your reaction mixture. It will work with you. Please check a small scale reaction and find if it is working?
I have tried 2.5% DMSO in the reaction,but it doesn't work every time!Can you give me some other advice?
Dear Mao,
are you sole your problem about GC-Rich real time PCR? I also meet this problem now, could you give me some advice ? thank you !
Normally, I redesigned primers for QPCR to avoid the GC-rich area. The sizes of PCR products are best at 48-145 bp if, say, using SYBR Green Master Mixes from Invitrogen which can also allow you applification of up to 75% GC content. It is not that hard to pick up the right primers in my case.
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