reverse transcripatase in Taq?
svetlov at oncology.wisc.edu
Mon Oct 20 18:08:05 EST 1997
In article <3447D881.7FB1 at ubaclu.unibas.ch>, schuchert at ubaclu.unibas.ch wrote:
> It was once reported that Taq can also reverse transcribe RNA.
> What's the actual opinion on that?
Taq activity as RT in its "normal" (Mg++-based) buffer is negligible. Tth
has a substantial activity, but in Mn++-substituted system.
> I try to make rt-PCR of a weakly expressed gene and get specific bands
> even without reverse transcription (contamination can be excluded,
> the RNA has been treated with DNAseI and the genomic copy has an intron,
> so that can be excluded too).
> Any hints before I go and try RNAse treatment (reluctantly!!)
Increase the amount of material (template) 10 fold in your Taq reaction and
see if you get 10 times as much "contaminating bands" - that will tell you
whether you are getting your contamination with the template or from
elsewhere. Try different enzyme for the PCR amplification - like Tli or
Vent, that was reported to have no RT activity at all. BTW presence of
"specific" (you did not actually sequenced those, did you?) bands on RT-PCR
EtBr gels is not such a rare occurence - without RT or even without
template at all. In some complicated cases people add internal control - a
measured amount of the target RNA produced in vitro (SP6 or T7). Hey, PCR
is not food - you can play with it!
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