Inverse PCR

Chunxin Wang cxwang at wam.umd.edu
Fri Mar 2 09:19:34 EST 2001


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

You'd better do this step. My experience is that you may omit the phenol
extraction, just inactivate the RE by heat and precipitate with ETOH.

> 2) what is the optimal concetration of gDNA forformation of closed
> circles  (using plant dicot gDNA)

1 ug gDNA is optimal for Arabidopsis for digestion, then dilute 200-500
times for ligation. I found that ligation at 14C for O.N. is much better
than at RT.

> 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)?

I prefer not use blunt cutters as the ligation efficiency is lower.

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

I don't think nester primers are necessary.

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

All depends on your primer. Use same concentration of primers as usual.
 
hope this help.
Black


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