PCR with E. Coli

ANSIDHP at EARN.OSUCC ANSIDHP at EARN.OSUCC
Fri Sep 4 07:44:00 EST 1992


Hello Netters,
I routinely perform PCR to amplify short (100-500) bp regions
of mammalian DNA.
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? I would think that the concentration of genomic template
might be reduced, due to the smaller genome size.
Also, are there some tricks for amplifying longer regions of DNA?
I normally use 1 minute time spans for all 3 steps in a cycle.
Should the extension interval be lengthened?
Any helpful comments would be greatly appreciated!
Daniel Pomp
Department of Animal Science
Oklahoma State University
ANSIDHP at OSUCC
(405)744-6070 (Tel)
(405)744-5747 (Fax)



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