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differential precipitation of PCR

Steven L. Sabol sabol at codon.nih.gov
Thu Jul 14 21:35:22 EST 1994


Kenneth M. Pfarr (kpfarr at Emerald. tufts.edu) wrote:

:: 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! 

Does anyone know whether this protocol works when the PCR primers are
labeled with biotin or with digoxigenin?

Suping Zhang
Department of Microbiology
Uniformed Services Univ. of the Health Sciences
Internet: szhang at usuhs.usuhs.mil





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