"Miller, Mark J. (NCI)" wrote:
>
> Dear ABRF'ers:
>
> Theoretically, I would think that a primer used for PCR should double as a
> sequencing primer, provided one has cleaned the PCR product completely from the
> two primers. However, frequently it does not. One of our users has some BACs
> from which he's PCRed up a fragment. After gel purification and extraction from
> the gel, he's tried to use the PCR primers for sequencing and has generally
> failed miserably. No sequence at all (though there is a "T" blob at ~110 bp -
> now what is the cause of that?). Any general procedures - or references thereto
> - for sequencing PCR products will be appreciated. Thanks,
Mark,
You don't say whether BOTH primers give blank lanes ...
One common problem is if only ONE of the PCR primers is actually
performing the amplification - at both ends. In other words, it's not
the legitimate PCR product. Symptom of this will be that one of the two
PCR primers gives a blank lane in sequencing, while the other primer
gives two superimposed sequences (peaks-on-peaks). Make sure they're
verifying the identity of the PCR product appropriately.
Another thing to check is the concentration of the template. I have
often had to explain to clients that there's not enough DNA in most PCR
reactions to run an OD (unless it's a big reaction or a micro-volume
spectrophotometer). If you were not the one who prepped the fragment,
ask them all the pointed questions about the isolation - did they
quantitate the DNA *after* the gel isolation? Was the amount recovered
realistic based on the size of the PCR reaction and subsequent steps?
(and as always, tell them to double-check calculations).
Good luck -
Bob Lyons
University of Michigan
-----------------------------
Robert Lyons, Ph.D.
Director, DNA Sequencing Core
University of Michigan
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