Method for Anchored PCR/RACE

George W Chacko gchacko at magnus.acs.ohio-state.edu
Thu Oct 15 09:35:46 EST 1992


In article <1992Oct15.162328.123 at chmeds.ac.nz> cytogen at chmeds.ac.nz (Martin Kennedy) writes:

> Finally, anchored PCR doesn't seem to work over regions much longer
> than about 500bp, at least in my experience. Some PCR reactions show
> a nice tight band, others a smear - 

I've gotten tight bands upto 1 Kb using a 3' RACE system. I use 2 ug
of total RNA (the quality of RNA appears to be the most critical
factor IMO). I reverse transcribe using the Pharmacia cDNA synthesis
kit adding 1ug of their supplied Not1-dT18 primer in a 15ul reaction
as described in their manual. 37C for 1 hour and I amplify 1 ul of the
RT product in a 100 ul PCR reaction. The downstream primer I use is
identical to the linker region between the dT and the Not1 site. The
upstream primer I use is message specific. I also use a touchdown PCR
protocol starting with annealing at 65C and dropping 0.5C until 53C
with a total of 40 cycles. I Elutip the PCR product and clone it into
a T-vector which we make in the lab and it seems to work. The
touchdown PCR seems to have cleaned up the smears I used to see
earlier. 

George



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