mysterious PCR question

Dr. James J. Campanella campanellj at mail.montclair.edu
Wed Jul 18 12:56:01 EST 2001


Hi Warren,

Thanks for the response. I appreciate it.

Here's the telling experiment. I diluted the DNA template by 1:10, 1:100,
and 1:1000 and still got a big smear in each instance when I performed the
PCR. I was really hoping this would work, but I was a bit surprised when it
did not. I was amazed that I even got any amplification at all at the
1:1000 dilution.

Did I not dilute out enough? Any other suggestions or thoughts?

Jim

At 04:28 PM 7/17/2001 -0600, Warren Gallin wrote:
>James,
>	Sometimes I get a result like this with too much template.  I've
>usually rationalized this as having a lot of template early that will
>self-anneal out of register at low probability and the be amplified.
>	Anyway, when I am reamplifying I do a set of serial 10-fold dilutions
>of template and usually get one or two reactions that have the desired
>band.  Your mileage may vary.
>
>Warren Gallin
>
>"Dr. James J. Campanella" wrote:
>> 
>> Hi everyone,
>> 
>> I've got a quick PCR question. I just amplified a cDNA of a
>> gene of interest using RT-PCR. I gel purified the cDNA and
>> isolated it using Gibco-BRL's gel DNA extraction kit.
>> There was not much cDNA from the RT-PCR so I took the
>> purified DNA and amplified it by an additional PCR rxn
>> using primers known to amplify the sequence.
>> 
>> When I ran it out on a gel, I got a huge amount of DNA
>> from the rxn, but it was a high molecular weight smear
>> from about where the cDNA would have been at about 1400 bp
>> up to perhaps 8000 bp. Yes, a very big smear.
>
>

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