How much RT primer do I need?

[John Dixon]

Hi, I have been trying to do RT-PCR using total RNA extracted from murine
ES cells. So far I have been having all sorts of problems with RNAses, but
I have just noticed a figure in my protocol that makes me suspicious.

The original version was designed for random priming using hexamers, and
required 13.5ug of primers. I didn't notice that this was for hexamer
primed synthesis, and used 13.5ug as the figure to aim for with my RT
primer. However I am using a 65mer with 14 T residues 3' of a 34bp loxp
site and with a 19bp 5' clamp sequence for priming the PCR. This is
20ug/nmol and I am wondering how much I should use in each RT reaction
(10ug tot RNA in 75ul reaction vol).

After picking a few randomish hexamers I get an average of 1.8ug/nmol for
the hexamers so 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?

Please tell me I need less primer for dT primed RT (as 65mer's dont come cheap!)

Can anyone deconfuse me?

-- 
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