In article <2vrp7o$etk at usenet.INS.CWRU.Edu>,
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.
To a 100-ul PCR reaction, add 50 ul of 7.5 M NH4OAc and 150 ul of cold
100% ethanol. Mix gently and let it sit for 5 minutes at room
temperature. Spin in a microcentrifuge for 15 minutes. Remove the
liquid, wash the pellet with 70% ethanol, dry and resuspend.
This procedure removes unincorporated primers and nucleotides well enough
that the DNA can be used for sequencing. We have used it on PCR products
that are 300 bp and larger; I don't know how much smaller you can go
before the DNA doesn't precipitate.
John H. McDonald
Department of Biology
University of Delaware