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Selective PCR amplification

Harold Rees hrees at mail2.sas.upenn.edu
Thu Oct 24 09:25:27 EST 1996

Doug Johnson (djohnson at OREO.CHEM.UOTTAWA.CA) wrote:
: We are trying to clone several closely related sequences via PCR 
: using the same primers. The products are different sizes, from 300 to 
: 600 bp. When we amplify and clone, we have to screen many recombinant 
: before we can find the smaller ones. Is it possible to control the PCR 
: conditions e.g., by lowering the time of extension, in order to selectively 
: amplify the smaller products?

Try running your PCR product on a gel (using TAE with low EDTA 
concentration).  Then, cut out the band you are interested in, melt it, 
and use some in another PCR.  Doing this should allow you to amplify 
mostly the cut-out sequence, thereby reducing the amount of larger 
products.  You may have to do it more than once.

Hal Rees                                ********************************
Monell Chemical Senses Center           *"Oh well, whatever, nevermind"*
Philadelphia, PA                        *  -Kurt Cobain                * 
hrees at mail.sas.upenn.edu                ********************************

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