An RT-PCR problem to sink your teeth into
Gregory S. Buzard 'Buz'
buzardg at MAIL.NCIFCRF.GOV
Wed Sep 4 16:20:24 EST 1996
Katheryne Coyne wrote:
Subject: Re: An RT-PCR problem to sink your teeth into
OK, Bionetters, here's a problem you can sink your teeth
into: I'm doing RT-PCR on mussel larvae total RNA to
determine at what developmental stages a particular gene is
Here's where things get strange: Both my positive control
RNA and my standard RNA without the deletion gave a very
weak band (barely visible) of the correct size and two even
weaker smeary bands that were smaller by 100-200 bp.
At this point, I know my primers are no good, but why? Can
anyone give me a good explanation as to why the RNA
template works ok with these primers if a small section has
been cut out, but not if left intact?
I agree with the respondents that you already have. The
deleted control may have lost a strong secondary structure
that inhibits the PCR. The weak bands you see below the
band you want may be single stranded DNA, if you are
analyzing by agarose, and primer dimer from a frustrated
Try lengthening you extension time during cycling, and give
the final product 10 min at 30 degrees before a terminal 72
degrees to 'polish' the product.
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