RT-PCR of bacterial RNA

kertesz at micro.biol.ethz.ch kertesz at micro.biol.ethz.ch
Thu Jun 15 18:36:20 EST 1995


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




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