Does anyone have an experience in preamplification of samples before running real-time PCR? Repeatedly, I don't get enough cDNA for efficient real-time PCR amplification. Would it be possible to amplify in regular PCR for some 30 cycles and than to use the resulting solution as a template for real-time PCR? Also, would it be worthy to try to amplify in more than 40 cycles of real-time PCR? What are the limitations? I'm not experienced in molecular biology, so if I make some obvious mistake in the reasoning, please don't hesitate to explain me the basics.
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pcrman
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Posted 21 May 2009 - 07:48 PM
Hi Georgije,
It is not a bad idea to preamplify before real-time PCR. As a rule of thumb, I don't use more than 36 cycles for RT-PCR. Any amplification after 36 cycles is not real. If you have enough RNA, the best thing to do is to generate enough cDNA by RT reaction.
It is not a bad idea to preamplify before real-time PCR. As a rule of thumb, I don't use more than 36 cycles for RT-PCR. Any amplification after 36 cycles is not real. If you have enough RNA, the best thing to do is to generate enough cDNA by RT reaction.
