Cloning problems-supercoil

Dr. Duncan Clark duncan at genesys.demon.co.uk
Tue Sep 9 10:05:02 EST 1997


In article <lvisser.1.000E965C at scientia.up.ac.za>,
lvisser at scientia.up.ac.za writes
>My big problem at the moment is to try and sequence the 1400 bp insert. I have 
>tried to denature the super-supercoiled recombinant plasmid at 96 degrees for 
>5 min. as well as alkaline denaturation as used for manual sequencing. Both 
>these gave very weak base signalling with the ABI sequencing, as if too little 
>template was present.  


I remeber seeing a apeper in NAR which suggested, that with severe AT
rich DNA, one should lower the extension step in PCR from 70C to 60C in
order for it to work. It could be that in your cycle sequencing your DNA
never extends because it is above the denaturation temp. for 80% A+T
rather than because you can never denature the plasmid. So try
sequencing at 60C.

Duncan 
-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
TEl/FAX 01252376288
http://www.dnamp.com
http://www.genesys.demon.co.uk



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