Inverse-PCR of long DNA

ww40 William_D_WARREN at UMAIL.UMD.EDU
Fri May 13 13:34:15 EST 1994


>dear netters,
>
>we're trying to do Inverse-PCR with a approx. 4.5 kb fragment (estimated from
>Southern-blot). As we remember there was some talk about how to amplify long
>DNA-fragments. If anyone has done Invers-PCR of such length or has any other
>hints - please post. 
>

I've amplified inverse PCR fragments from Drosophila  and housefly genomic
DNA of the length you are interested in using straight Taq polymerase and
long extension times (up to 6 minutes). Although I managed on one occasion to
do it with a single pair of primers and around 40 cycles, I now routinely use
two pairs of nested primers.

BILL


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William D. WARREN, PhD
Center for Agricultural Biotechnology     Email: ww40 at umail.umd.edu
University of Maryland at College Park    Phone: (301) 405-7681 

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