Direct sequencing of lambda phage dna

RichWalko richwalko at aol.com
Fri Nov 4 09:57:00 EST 1994


In article <1994Nov2.144926.27119 at oxvaxd>, rmewing at vax.oxford.ac.uk
writes:

>Anyone have experience with direct sequencing of lambda phage DNA?


Lambda sequences reasonably well using cycle sequencing, either manually
or on an automated sequencer.  Many companies produce cycle sequencing
kits for manual radioactive sequencing and generally recommend
end-labelled primers (32P)be used, though some may give resonable results
with 35S-dATP incorporation.  Check the instructions with the kits for
their specific recommendations on label and amount of DNA (usually a few
(1-3) micrograms is required.)  The results are generally acceptable for
checking the ends of clones, screening, short (200 or so bases manually)
reads when you are trying to avoid the trouble of cloning.  If you are
looking for clean, error-free, long reads on a whole long clone, you are
probably better off just subcloning into a plasmid vector and using that
for sequencing.  I think the results are overall better quality and your
read lengths should be longer, but it's obviously more work.

Claimer/Disclaimer:  Just so there is no misunderstanding, I work for
Pharmacia Biotech.  We do sell cycle sequencing kits for both manual
sequencing and our ALF automated sequencer.  And, yes, they will work for
your application, but so do other companies.  My statements here and above
are not meant to be a sales pitch, but my experience from working with the
ALF.  I recommend you check with your local mol bio company sales reps,
try several companies' kits, and decide if the results are satisfactory
for your needs, and which kit is best for you.  



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