Inverse PCR Help

Rob Allen r.allen at pi.csiro.au
Wed Feb 28 18:49:31 EST 2001


I have a 1.7Kb partial plant cDNA. I was thinking of using Inverse gDNA
PCR to retrieve ~400 b.p from the 5' end. I was going to use nested
primers and a several different 6 base cutters- I guess this is like
shooting in the dark but most 4 base cutters cut inside my sequence. The
other option is to use several blunt cutters, or end filling- any
ideas?. Also the concentratrartion of gDNA I guess must be low to favour
recircularisation- anyone have definate concentrations which work with
dicot DNA. The other problem is that Im working off a cDNA to design
primers for gDNA which may infact span introns- I guess testing them
first on gDNA (i.e making complementary primers to the nested primers)
would help but what about consensus sequences in the cDNA which may give
clues to introns? Finaly some people say that after digestion Ethanol
precpitation is only required prior to ligation while others say heat
inactivation or phenol/chloroform extration is needed as well. Thanks in
advance.

Rob Allen
CSIRO Plant Industry
Canberra Australia


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