PCR troubleshooting (fwd)

Terese M Barta-1 barta001 at STAFF.TC.UMN.EDU
Thu Jul 8 17:08:12 EST 1993


Thanks, Andre, for the answer to my query:
(T. Barta, U of MN)


---------- Forwarded message ----------
Date: Fri, 2 Jul 93 10:04:29 CDT
From: hamel at ccu.UManitoba.CA
To: barta001 at staff.tc.umn.edu
Subject: Re: PCR troubleshooting


YES ... RNA can/will adversely affect PCR ... too much RNA will inhibit
RNA as well as DNA PCR.

Treat few ug of DNA with RNase A for 30 minutes at room temp then
phenol/CHCl3 ext'ns. ... can also alk denature (0.2 N NaOH final conc)
at 37C for 10 min, then add either 0.3 M NaAc, pH 5 (final conc) or
2.5 M NH4Ac (final conc) then 3x vol ethanol ... mix, ppt -20C at least
few hours, spin, wash, etc ... RNA is destroyed by alk den and DNA is left
unharmed AND denatured into ss

cheers, Andre
                            ********************
Andre Hamel                                 email: hamel at ccu.umanitoba.ca
Manitoba Veterinary Services                lab tel.: (204) 945-7630
Infectious & Genetic Disease                     FAX: (204) 945-8062     
Mol.Biol.Lab., Winnipeg, Manitoba, CANADA
                            ********************





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