trouble with RT-PCR

Peter Ellis pjie2 at cam.ac.uk
Thu Mar 23 18:19:55 EST 2006


Sanni wrote:
>
> I also checked the sequence for retroposons,
> but could not find any!

That doesn't mean there aren't any.  :-)

>
> Is it somehow  possible that the specific PCR
> primers could bind to anbundantly expressed mRNAs???

Not really.

>
> Any hints or tips would be appreciated ;-)

Forget the RT part for now - just do a standard PCR with genomic DNA 
template, see if you get both bands.  Try cutting and sequencing the bands 
to check whether your primers are contaminated or mispriming.  Once you know 
your primers are giving you what you expect, then go back to doing an 
RT-PCR.  Doing a DNase digest on your RNA sample before you do the RT 
reaction will prevent problems with genomic contamination in almost all 
cases.

Peter Ellis 




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