RT-PCR of bacterial RNA

John W. Morgan John_Morgan at brown.edu
Fri Jun 16 09:33:21 EST 1995


  A tip that may come in handy for you:  I was getting mixed results from
my RT-PCR; some samples worked fine, some totally flopped.  Then I stopped
using previously _frozen_ first strand cDNA samples.  Viola!.. every pcr
product worked just fine.  Moral:  try using only "fresh" first strand
samples; i.e. perform the first strand synthesis and pcr amplification the
same day, without freezing.
  Hope this helps.




In article <kertesz.8.00129B94 at micro.biol.ethz.ch>,
kertesz at micro.biol.ethz.ch wrote:

> I am trying to detect a rare transcript from Pseudomonas aeruginosa using 
> RT-PCR, but not having much luck so far. The RNA seems to be ok (nice sharp 
> rRNA bands on an agarose gel), but so far I haven't been able to find any 
> evidence for my transcript, despite the fact that the corresponding enzyme is 
> present in the cell extracts (harvested simultaneously with RNA preparation) 
> in a highly active state. The two primers I'm  using should give a PCR 
> fragment of 800 bp (in fact, they DO give an 800 bp fragment with control 
> DNA!). Is 800 bp too long for the reverse transcriptase to deal with 
> efficiently without falling off? I'm heating the reverse primer with the RNA 
> at 65°C as described in the Big Red Book before doing the RT step.
> 
>  Or is my transcript just too unstable? (I haven't been able to see it by 
> Northern either - not yet anyway).
> 
> All tips gratefully accepted!
> 
> Michael Kertesz
> 
> ETH-Microbiology,                                    
> CH-8092 Zurich, Switzerland.
> 
> Tel: +41-1-6323357     Fax: +41-1-6321148
> e-mail: kertesz at micro.biol.ethz.ch

-- 
John W. Morgan
Dept. of Pathology..........Roger Williams Medical Center
BioMed Dept.................Brown University
825 Chalkston Ave.          Internet:  John_Morgan at brown.edu
Providence, R.I.            telephone: (401) 456-6568
02908                       fax:       (401) 456-6569



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