template preparation for cycle sequencing

A.Marchant marchaa at agric.nsw.gov.au
Wed Feb 22 19:48:57 EST 1995


I have been trying to sequence PCR products using two Promega kits, the
'Silver Sequence' system, and the 'f-mol' system.  Both of these use
cycles of annealing and extension, and strand separation, and are done in
a PCR machine.  I started with the silver sequence system, and got signal
too low to be readable.  I tried upping the template concentration, and
could see some faint sequence bands (not readable) in the +ve control (their
supplied plasmid DNA).

I then switched to their f-mol system, which is exactly the same, except
you end-label the sequencing primer with 32P.  This should have given me
much more signal, and so give me a better idea of what's going wrong (my
intention is to go back to silver staining when it is all debugged).

My results:  no sequence; there are faint bands which presumably represent
full replication products of the templates, nothing in the middle of the
gel, and a lot of bands at the bottom of the gel, which mostly are in all
lanes of all templates (in fact, the X-ray, turned upsidedown, looks like
quite a successful PFGE!).  There is a faint background throughout every lane.

I have not done anything special to the primers, except for
5'-end-labelling them with 32P, nor have I purified the template (PCR
product) in any way. What should I be doing?  The PCR-product-template is
only a small component of the sequencing reaction, so I thought all the
little molecules in it wouldn't matter (and they aren't labelled). 
If I should be doing something to the primer, what is it, and why?

Thanks for any advice.  Please E-mail.

Adam Marchant
marchaa at agric.nsw.gov.au






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