problems with PCR

Winoto lab lab_winoto at maillink.berkeley.edu.
Tue Jun 28 19:23:39 EST 1994


In article <2u4ed7$lr2 at mserv1.dl.ac.uk>
WMGDDM <WMGDDM at CARDIFF.AC.UK> writes:

> I've got a problem with a PCR that I'm attempting...
> 
>     I'm trying to use a pair of primers to follow a series 
> of deletions that I'm making in a plasmid.
>     
>     Under 'standard' conditions I can amplify the deleted 
> version of the plasmid but not the original undeleted 
> version.
> 
>     Can anyone explain this, I've tried all the usual 
> variations in the reaction conditions, etc. with no luck

Well your description of your your reaction conditions is pretty vague
so it might be hard to answer this question.  For instance have you
ever been able to use these primers on the original plasmid?  Are there
differences between the quality of these preparations?.  Try to fix
what usually is the most common problem when amplifying from plasmids. 
Some plasmids are too tightly supercoiled for given primers to anneal
efficiently.  Try linerarizing both preparations and then try your
amplification.  This will allow efficient hybridizations and will also
insure that both DNA preps are in identical conditions.

Pendragon 



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