Inverse PCR

Rob Allen r.allen at pi.csiro.au
Thu Mar 1 19:53:38 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 forformation 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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