Cycle sequencing /colony sequencing

Rene r.j.nederhand at st.hanze.nl
Fri Jun 27 17:23:13 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



More information about the Methods mailing list