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
> 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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