PCR with very low DNA concentrations

Duncan Clark Duncan at genesys.demon.co.uk
Mon Jun 19 10:57:59 EST 1995


In article: <95167.151043MDC128 at psuvm.psu.edu>  Lord Antarchon <MDC128 at psuvm.psu.edu> writes:
> 
> Is there a established strategy one follows when trying to get PCR
> product from low concentration DNA, i.e., you increase DNA concentration
> first, then primers, etc. I am trying to amplify a low-expression gene
> from a lambda gt11-based cDNA library.  If anyone has a clue, please
> email mdc128 at psuvm.psu.edu, because I'm starting to get a bit vexed with
> my reactions.

The PE catalog gives some details that may help you ie

To amplify 500bp from lambda. Start with 1ng lambda ie 10pg of 500bp target or
1.81 x10E7 molecules. After 25 cycles one should get at least 10E5 fold 
amplification ie 1ug of 500bp product and 1.81 x 10E12 molecules. You should
be able to get some idea as to how much template (pfu) you will need to start
with. I would set up various PCR's with increasing template from 1ng 
(~2 x 10E7 Pfu) up and 50pM each primer per 50ul PCR. But first of all can
you amplify your gene from the cDNA used to make the library. If not it is
unlikely to amplify from the library. If that is OK you may have to play 
with annealing temp or Mg2+.

Good luck

Duncan

    

 
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