SOS Call to all experinced "Sequencers"

Michael DiDonato michael.didonato at utoronto.ca
Thu Mar 28 16:42:43 EST 1996


> Please,I know sequencing is not easy and may be take ages, but I just need
> to know which would be to best method and time saving. My bands are
> 200bp,350bp and 600bps long.
> 
> Thanks a million.
> 
> My e-mail : spyong at pc.jaring.my
> 
> Cheers,
> Stephanie
> 
The easiest way is to subclone into a PCR cloning vector such as the T/A
cloning system from clonetech.  The reason for this is that if you try to
sequence the product directly you will miss a chunk of sequence right
after the primer (about 40 bp).  By cloning into the vector first you
could use a primer in the vector and sequence into the insert.  The
advantage with the vector is that you can grow it in large quantities and
'cut' out your insert later.

Alternatively you could sequence the fragments directly, but you would
have to sequence in both direction to get all the sequence at both ends.

Sequenase is just a sequencing kit you can buy to make your life easier (I
forget who sells it).  Pharmacia also sells a T7 sequencing kit which is
very good.

Hope this helps.

Michael

---------------
Michael DiDonato
Department of Biochemistry
University of Toronto
michael.didonato at utoronto.ca



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