mRNA->cDNA->PCR

gagnon at vax.ox.ac.uk gagnon at vax.ox.ac.uk
Fri Feb 26 04:06:54 EST 1993


Dear Art,
There is one more protocol that may be of interest if you are facing many RNA 
preps from tissue culture cells. It is described in detail by N.M. Gough in 
Analytical Biochemistry, v.173, pp.93-95 (1988). It takes only 30 mins to 1 
hour to complete and is based on the mild lysis of cells with NP-40, followed
by extraction of proteins with SDS/urea and precipitation.
I have used it twice for cloning the cDNA of antibody variable domains from 
hybridomas and it worked well in both cases. From 5*10^7 cells I recoverd 
around 500 micrograms of total RNA (OD260/OD280=1.95) (by using ten times scaled-
up version of the original protocol). I used 10 micrograms of this RNA as a 
template to synthesize the first strand in 20 microliter volume with Amersham RT 
kit, priming with oligo-dT. 0.1 microliters of this reaction mix was more than
enough for a PCR reaction.

With best wishes
	Armin Sepp    



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