RT-PCR HEEELP!

Karen Allendoerfer ravena at cco.caltech.edu
Mon Mar 24 20:50:56 EST 1997


In article <01bc3895$b97275a0$LocalHost at marta>,
Marta Radman-Livaja <marta.radman-liavja at zg.tel.hr> wrote:
>
>I am trying to amplify the 5HT-2A mRNA from total rat platelet RNA with the
>RT-PCR method. I have just once obtained a faint band on EtBr agarose gel.
>Even though I used the same protocol for other samples, my labor has been
>in vain. 
>Can anyone who succeeded more than once in a similar experiment contact me
>and give me a few tips. I'd be very grateful 

I've had better luck with polyA+ mRNA as template than with total RNA.
With total RNA if you're using degenerate primers, especially (as I was),
you may be more likely to pull out some ribosomal gene instead of
what you want.  

Also, if your results are variable, look at the bottom of your lanes--
is there a diffuse band there?  That's probably primer-dimer, and can
be minimized by doing a "hot start" with either the taq or the mg in
wax beads.  Or, if the wax beads aren't available, heat the samples up
to 60-80 degrees before adding the taq.  

Finally, it's possible that one round of amplification isn't enough to
see your band.  Try taking the reactions that you have and using THEM
as template for another round of amplification.  This may bring your
signal up to the visible level on an EtBr gel.

Hope these suggestions are useful to you!

Sincerely,

Karen Allendoerfer


>





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