Summary for "Non-specificity for Primer Extension?"

Martin Leach leach at mbcrr.harvard.edu
Tue Apr 5 07:50:25 EST 1994


In article <2ns1t6$4ke at usenet.INS.CWRU.Edu>, bl275 at cleveland.Freenet.Edu
(Dan Diaz) wrote:

> 
> In a previous article, smrodems at students.wisc.edu (Steve Rodems) says:
> 
> >I believe actinomycin D is an inhibitor of RNA polymerase II.  This is
> >included so that any RNA products synthesized in the tube are derived from
> >the RT and not from RNA polymerase.  I don't know where the RNAP II would
> >be coming from since most protocols call for guanadinium treatment to
> >release RNA which I believe has its way with most proteins.
> 
> actinomycin D inhibits second strand synthesis during first strand cDNA
> synthesis by reverse transcriptase.  it is typically used with MMLV RT,
> whereas pyrophosphate is typically used with AMV RT, as MMLV RT doesnt work
> well in the presence of pyrophosphate.
> 
> diz


Forgot to mention a couple of things from the original posting.


1. Try a total RNA source that lacks the message of interest using the same
RT primer

eg. if u r looking at a human brain message - use human hela cell RNA as a
negative control (in case your primer sticks to some ubiquitous message)

2. A good way to check your PE products is to transfect your promoter
reporter construct into cultures, isolate the total RNA and do PE using a
primer which sticks to your reporter gene.


I'm doing the above at the mo. to check my PE.

Any comments (scientific regarding PE)

Would greatly be appreciated.

BTW. Tried the actinomycin D method recently - saw no bands everything
disappeared and didnt confirm anything :(

Martin
-- 

.....          Martin Leach                Email:leach at mbcrr.harvard.edu 
   _|____      Dept. of Pharmacology       Phone: (617) 638-5323        
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