Mark,
As you say, most of the time it will work. But, think of all the bizarre small bands that can be generated in a PCR reaction and picture them not as distinct bands on the bottom of the gel (or run off) but as a bright comet with a lingering tail that trails all the way back to the gel slot. Now when you cut that PCR band out you pick up a small amount of the "tail" from primer dimer bands etc. By weight they don't amount to much at all (can't see 'em) but the molar amount is high being that they are so small. These will blob up your sequencing because they all contain the PCR primer sequence.
Better to design a new primer by moving in just a few unique nucleotides into the fragment.
Or better yet just sequence the BAC directly, (if that is all they want).
Reference: Somewhere there is a paper about the distribution of a sample run through a matrix with an electric field. My brain can't handle all the statistical formulas so I fall back on Stephen Jay Gould explaining why he is still alive. His prognosis was similar to the gel band, "highly right skewed asymetrical distribution"
His explanation: http://cancerguide.org/median_not_msg.html
hope this helps, Paul
____________________________________
Paul Morrison JFB216 paul_morrison@dfci.harvard.edu
Molecular Biology Core Facilities
Dana-Farber Cancer Institute http://mbcf.dfci.harvard.edu
44 Binney Street 617-632-3082
Boston, MA 02115 fax 632-4814
____________________________________
On Tuesday, March 28, 1939, Miller, Mark J. <millerm@dc37a.nci.nih.gov> 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 J. Miller, Ph.D.
>Manager: DNA Sequencing MiniCore Facility
>Bldg 37, Rm 3C28, MSC 4255
>DBS/NCI/NIH
>Bethesda, MD 20817-4255
>
>301-496-5688 x226
>mark_james_miller@nih.gov
>
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