An RT-PCR problem to sink your teeth into

[Gregory S. Buzard 'Buz']

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 
transcribed.
 
//SNIP///

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? 

Kathryn,
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 
Taq.

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. 

Buz
Gregory Buzard