3'RACE PCR

John Watson watson_j at bms.com
Wed Feb 19 16:34:27 EST 1997


czlq at musica.mcgill.ca wrote:
> 
> Hi,I am doing 3'RACE  PCR in the purpose to amplify  the  new sequence from the 3' end. I have successfuly amplified a band
> which looks like the  PCR product. After that I used several gene specific primers combing with the universal primer for further
> PCR in order to know what the PCR product is. The PCR were worked and I got single band from each PCR reactions with
> respected size. Sequence reaction proved that this PCR product was a combination of known sequence and the unknown one.
> My question is how can trust this result? Whether it is possible that DNA amplification can jump from one template to another duing
> the process of the PCR because for some unknown reason, PCR sometimes gives us the weild result.Thank you for your help.

Confirm the result by PCR'ing across the entire cDNA (amino to carboxy terminal 
ends).  This will tell you that your new sequence is contiguous with previously 
known 5' end.  Also if you use a proof reading ploymerse to do the new PCR 
reactions, you will also get an uncorrupted "good" sequence.

Cheers,

AJW
----------------
John Watson
Bristol-Myers Squibb Co.
watson_j at bms.com
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