Help in PCR sequencing

[Karl Voss]

Mr. J. Membrillo-Hernandez (jmembril at hgmp.mrc.ac.uk) wrote:
> Hello again,
> 	Has anybody of you tried to sequence a gene directly from a 
> colony?, any help would be gratly appreciated. Thanks

> 					Jorge

> 					University of London

> 					jmembril at iron.hgmp.mrc.ac.uk


Sure.

We have primers that bind in the region outside the multiple cloning site
in bluescript (I think that this is the B-gal gene).  We touch a colony 
with a pipet tip, squirt it into water (10ul) and boil 5 minutes.  Then
we do pcr with these primers and 1ul of the boiled cells.  Usually you see
only one band on the gel.  We do cycle sequencing directly on this pcr
product using about 1 - 4 ul of the pcr as template.  The ammount depends
on how well the pcr worked.  

This usually works pretty well.  Please contact me if you want any other
details.  I don't do this much myself but can ask the person in our lab
who does.

Karl

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Karl Voss
Department of Chemistry
University of Alberta
Edmonton, Alberta, Canada
T6G 2G2

karl at hobbes.chem.ualberta.ca
phone 403-492-0222
fax   403-492-8231