SOS Call to all experinced "Sequencers"

Leslie Jones ljones at dna.bchs.uh.edu
Thu Apr 4 16:15:53 EST 1996


In article <199603281727.BAA03464 at relay3.jaring.my>, spyong at pc.jaring.my wrote:

> Hello,,
> 
> The only sequencing I've ever done is through the text book. However, it
> seems like now is the time to start sequencing. I have three PCR products
> and need to sequence them before I work further only to find that they are
> junk. Someone suggested end-labbeling, others say double strand sequencing,
> some say cycle sequencing....some say stick to 'Sequanase' (whatever that
> means). Do I have to subclone into M13?
> 
> 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
> 
 
My best advice is to subclone into a sequencing vector such as pCRII
Vector (TA vector) from Invitrogen Corp.  It would be difficult to
sequence very short PCR products directly, especially if you don't have
any sequencing experience.  I was at a core facility at Texas A&M and the
kit that we used was SequiTherm Cycle Sequencing Kit by Epicentre.  This
is a thermostable polymerase based kit and is much easier to use than
Sequenase.

If you aren't experienced in sequencing, my advice would be to send it to
a core facility.  Typically, the cost of sequencing would include the
primers needed to sequence your clones.  If you did the sequencing
yourself, this is something that you would have to do.  Another thing to
consider would be contig analysis of your sequence.   This is another
service provided for by the core facility.

Hope this helps and good luck with your sequencing.



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