trouble with RT-PCR
pjie2 at cam.ac.uk
Thu Mar 23 18:19:55 EST 2006
> 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???
> 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
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