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differential precipitation of PCR

Kenneth M Pfarr kpfarr at Emerald.tufts.edu
Thu Jul 14 07:32:33 EST 1994


Seth Daniel Crosby (dd034 at cleveland.Freenet.Edu) wrote: 

: Anyone out there familiar with a protocol for the : differential
precipitation of PCR product from primers?  : By this I mean are there
certain EtOH/salt/temp : conditions in which large, double-stranded DNA :
fragments (the product) precipitate but small, single : stranded fragments
(the primers) do not precipitate?  : This would be cheaper and, I think,
faster than gel : and/or Wizard preps. : Seth Crosby

I have followed the protocol from Current Protocols in Molecular Biology
with great success.  Very simple:  add 7.5M ammonium acetate to a final
concentration of 2.5M.  Then add one volume of room temp. 100% EtOH and
sit at room temp. for 5 min.  Centrifuge for 5 min. and wash resulting
pellet with room temp. 70% EtOH.  Dry pellet and resuspend in appropriate
volume.  I used this method to remove excess primers when making template
for single primer reamplification of PCR product and for eventual
sequencing of said ssDNA.  Good luck! 




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