Best protocol to prepare BAC DNA

clive tregaskes clive.tregaskes at bbsrc.ac.uk
Fri Aug 28 11:18:42 EST 1998


Jun-Ichi Aikawa wrote:
> 
> I heve tried to prepare BAC DNA from 500ml E.coli culture using QIAGEN
> column and obtained 20ug DNA. When I cut it, looks like genomic DNA,
> showing no clear DNA digestion patten. HAve you ever prepared BAC DNA? What
> is best protocol?
> 
> Thanks.
> 
> Jun-ichi Aikawa, Ph.D.
> University of California, San Diego
We've had this problem in the past. Way we solved it was to grow up
several litres of bacteria, put the whole lot through a Qiagen column
and then put the resulting eluate through a caesium chloride gradient. 
We probably lose some of the plasmid through being nicked, but the
majority stays supercoiled and you normally get enough to see without
resorting to UV.

Clive Tregaskes
Institute for Animal Health
Compton UK
clive.tregaskes at bbsrc.ac.uk

The longer I work in science the more I believe in magic



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