colony sequencing question

ku at helix.nih.gov ku at helix.nih.gov
Thu Jun 26 13:49:39 EST 1997


R.J. Nederhand wrote:
> 
> I have just read the article about colony sequencing ('Colony
> Sequencing": Direct sequencing of plasmid DNA from bacterial colonies;
> Biotechniques 22:412-418,. March 1997).
> It looks like a very fast and relible way to sequence clones.
>  In the article the autors use the AmpliTaq cycle sequencing kit (Perkin-Elmer)
> for radioactive colony sequencing. I was wondering if it is possible to
> use normal Taq polymerase.
> I can imagine that 'normal' Taq will have problems with incorporating
> dideoxy nucleotides. Does somebody has experience with other
> polymerases?

"Normal" Taq does just fine with dideoxynucleotides. In fact a number of
sequencing kits use it. In particular we have used Taq to get sequence
from single colonies using a very simple procedure [Boil the colony in
TE add sequencing reagents and go (described in an article published in
NAR: Woodford, K. and Usdin, K. (1991) Nucleic Acids Res, 19(23), 6652].



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