Inverse PCR questions

"Rob Allen YD rallen at pi.csiro.au
Thu Mar 1 23:12:48 EST 2001


I had a few questions regarding inverse PCR:

1) Is it neccesarry to phenol chloroform or heat kill the restriction 
enzyme as well as ethanol precipitating prior to ligation?.

2) what is the optimal concetration of gDNA for formation of closed 
circles  (using plant dicot gDNA)

3) Is it better to use a range of blunt cutters, use 6 base cutters and 
end fill, or take pot luck with a series of sticky end 6 base cutters, or use 4 base 
cutters (most 4 base cutters seem to cut inside my known sequence)?

4) Im using nested primers, which have a few b.p overlap- is this o.k?

5) What cycling conditions are commonly used, and primer concentrations?

Any ideas or further suggestions greatly appreciated

Thanks

Rob Allen

CSIRO Plant Industry
Canberra, Australia



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