colony sequencing question

R.J. Nederhand RJ.Nederhand at pg.tno.nl
Thu Jun 26 13:53:53 EST 1997


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? 
I am also interested in the experience of other people using this 
technique.

This might be necessary:
The fragment I have cloned is 812 and 1133 bp large. It is ligated 
into a pMOS T-vector (Amersham) and after transformation in MOSblue 
cells (Amersham cloning kit) cultured on AXI-medium.

I look forward to your experiences.
Regards
Rene Nederhand
TNO Prevention and Health, Dept. of Vascular and Connective Tissue Research
Gaubius laboratory, Leiden
R.J. Nederhand
Zernikedreef 9
P.O. box 2215
2301 CE Leiden
The Netherlands



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