How much RT primer do I need?

John Dixon jpcd0 at mole.bio.cam.ac.uk
Wed Feb 1 10:39:52 EST 1995


In article <jpcd0-0102951527540001 at macr1-4.welc.cam.ac.uk>,
jpcd0 at mole.bio.cam.ac.uk (John Dixon) wrote:

> Hi, I have been trying to do RT-PCR using total RNA extracted from murine

[stuff deleted]

o they would be at 0.1mM in the final RT reaction. For the
> same molarity (0.1mM ie 7.5nm/75ul) I need 7.5x20=150ug of my RT primer.
> Surely this is crazy.
> 
> Are my calculations crap?


I have just thought of one reason why my calculation is crap...no make
that two: presumably the conc of hexamers has to allow for all the RNA to
be RTed not just the approx 1/50th which is polyA tailed mRNA, and of that
message only say 200bp of an average 3kb mRNA will be polyA potential
priming site.

So do I need 200/(3000*50) as much ie 0.01nM/75ul reaction? This would be
75 times LESS than what I am using now.

Aaaaarrrgghhhh- help -help 

Time for another tea-break, I think.

-- 
John Dixon                     Lab 44 (223) 334131
Wellcome/CRC Institute         Fax 44 (223) 334134
Dept Genetics
Cambridge University    
United Kingdom       e-m: jpcd0 at mole.bio.cam.ac.uk



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