Topoisomerase Use on BRL Supercoiled Ladder

Kim sunderme at u.arizona.edu
Wed Jun 18 16:45:12 EST 1997


Dear Netters:

I have been trying to clone a 10.3kb full length cDNA for some time now. 
My latest strategy is to make a gene specific library(Zap Express), do mass
in vivo excision, grow the mass transformation overnight, do the mass
mini-prep, load on a .6% agarose gel, cut out the 14-16kb bands, band prep,
transform and screen.

The 14-16kb bands yielded plasmids of 6-9kb.  I neglected  to think about
different levels of supercoiling.  I will now treat the BRL Supercoiled
Ladder and my plasmids of interest with topoisomerase I.  

Does anyone have any experience with this?  If so, please advise.

Thanks,
Kim Sundermeyer



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