Cosmid Sequencing

Martin Leach leach at mbcrr.harvard.edu
Wed Mar 30 12:27:26 EST 1994


In article <lyons-170394154246 at cyclid.demon.co.uk>,
lyons at cyclid.demon.co.uk (Alan Lyons) wrote:

> Does anyone have any advice on sequencing both Lambda and Cosmids on the
> ABI 373A? We have tried with a number of primers on Cosmids, some work OK,
> and some don't. We have had no success at all with Lambda. These have both
> been using Taq Dyedeoxy sequencing.


I managed to sequence Cosmid using the double stranded sequencing method a
couple of times.

First i digested 10ug of cosmid dna with a semi-frequence cutter, such as
pstI or EcorI

i then phenol extracted the dna and precicpitated it. Then made the usual
sequencing pellet by denaturation from this 10ug of template.

I managed to get novel sequence of this cosmid. Which i know is from the
gene i am interested in..l



(i just thought linearise and make a mixture of fragments, then let the
primer do the sorting for u.)

Martin.
-- 

.....          Martin Leach                Email:leach at mbcrr.harvard.edu 
   _|____      Dept. of Pharmacology       Phone: (617) 638-5323        
   / o  /      Boston Univ. School of Med. Fax:   (617) 638-4329         
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