*Reliable* RPA method?

Gary Kucera kucera~gt at glaxo.com
Wed Jul 17 07:16:56 EST 1996


>
>   After RNase treatment (in about 250 ul volume) adjust to 0.5% SDS and
>add 5 ul of 20 mg/ml Proteinase K. Incubate 15 minutes, 37oC. Do a single
>extraction with phenol-chloroform, then ethanol precipitate, adding
>appropriate amounts of NaAC, a little EDTA and a few ug of yeast tRNA as
>carrier. After centrifugation, remove supernant _completely_ (or do EtOH
>wash) and resuspend pellet in formamide containing gel loading mix.
>
>    
>
>   Hope this helps,
>
>      Pam Norton
>
>-- 
>Pamela A. Norton, Ph.D.          Assistant Professor of Medicine
>Thomas Jefferson University
>Philadelphia, PA 19107           p_norton at lac.jci.tju.edu



Pam,

Even easier is the following which I have used for the past 3 years 
with never a problem:

· T2 RNase Digestion 

Add 350 ICE COLD T2 RNase buffer (50mM NaOAc pH 4.5 2mM EDTA 100mM NaCl)

 containing 16 U/ml T2 RNase (BMB.) 

Incubate 1 hr. at 30oC 

Add 120 ul 5M NaOAc, 3 ul tRNA (20 mg/ml), 1 ml EtOH 

Ppt, then wash in 70% EtOH 

Resuspend in 8 ul Loading buffer 

Electrophores thru 6% denaturing polyacrylamide gel 

____________________________________
Gary T. Kucera, Scientist
Cellular Sciences/Transgenics Group
GlaxoWellcome Research, Inc.
Five Moore Drive
Research Triangle Park, NC 27709





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