PCR with E. Coli

Bruce Byrne byrne at soma.umdnj.edu
Fri Sep 4 13:07:56 EST 1992


ANSIDHP at EARN.OSUCC writes:
[stuff deleted]
>I am now about to help a colleague attempt to amplify a large
>(%2 kb) fragment in E. Coli.
>For mammalian DNA I normally use:
>100 ng genomic template
>300 nM each primer
>200 uM each dNTP
>1.5 mM MgCl
>in 50 ul reactions.
>Should I alter any of these conditions for amplification in
>E. Coli? 
[more stuff deleted]


Reduce DNA concentration by 10^3 to get same number of targets.  
High concentration of targets will kill your reaction.
Concentration of primers:targets will remain constant as
you are used to.  Your Mg concentration will need to be 
optimized to primer sequence.  Watch out for EDTA in DNA buffers.
Your Mg is at the low end of my experience.  Your 1min x 3 cycle
is probably a sufficient extension time and longer than necessary
for denaturation or annealing.  You may be cooking some taq
unnecessarily.

>Any helpful comments would be greatly appreciated!
     ^^^^^^^
     ...if it works, it was helpful.  Good luck.

Bruce Byrne


>Daniel Pomp
>Department of Animal Science
>Oklahoma State University



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