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HELP! How do you purify a 150bp PCR product for sequencing

Richard R. Hardy hardy at mighty.fccc.edu
Fri Dec 22 17:05:16 EST 1995


In Article <DJy1It.D6s at uns.bris.ac.uk>, Barry Vipond <vipond> wrote:

>I'm trying to purify a 150bp PCR product for automated sequencing. I've got
>a fair number of samples, so I don't want to gel purify. I've tried silica
>(Home-made), but the efficiency is very low (bigger products are fine). Glass
>fog or Mermaid, I assume will co-purify primers too.
>
>What do I do? Does anyone have any nice recipes/methods that they've tried
>under similar circumstances? Perhaps there is a better binding buffer for
>the silica - I was using Guanidinium Isothiocyanate.

If you have one STRONG band, then simple cleanup (PEG ppt or cleaning up
with spin column) may be OK.  ALternatives are to use the Qiagen, Amicon
or Wizard DNA microprep kits.  At least on ABI sequencers, glass binding
protocols are discouraged for PCR products.  We've noticed that carryover
of trace amounts of ethanol from the cleanup can severely degrade the
quality of the sequences.  Of course, carryover of trace amounts of ANY
organic/salt may also do this...  Good luck, many labs are doing this
routinely, so it is possible!
____________________________________________________________________________
Richard R. Hardy                       Member, Institute for Cancer Research           
Fox Chase Cancer Center                Tel:    (215) 728-2463
7701 Burholme Ave.                     FAX:    (215) 728-2412
Philadelphia, PA 19111                 E-MAIL: RR_HARDY at fccc.edu



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