Best protocol to prepare BAC DNA

Stephen R. Lasky srlasky at u.washington.edu
Mon Aug 31 18:41:44 EST 1998


I might suggest that you take Qiagen's advice on overloading their prep
columns seriously.  In my experience, with any type of vector, too much
input DNA does not make for good output DNA.

We usually use no more than 100 ml cultures for the midi columns when using
qiagen preps.  The most important step is the lysis step with careful.
mixing.  Very carefulllllll.  I mean, just roll the tube (a 50 ml conical)
around its short (not long) axis, 4 or five times.  More yields unacceptable
E.coli DNA contamination. we find that this will produce DNA that can be
mapped and subcloned and sequenced with low E.coli contamination (<10%
usually).

Our high throughput sequencing and bac-end facilities successfully use
Autogens for BAC DNA preps, making mappable, cloneable, directly
sequenceable DNA from 5 4ml tubes, but that is beyond most labs needs.

The bottom line, from our experience,  is that if you want to get a good
yield with low ecoli background, follow the qiagen bac protocol and do a
very gentle lysis.

hope that helps

srlasky

clive tregaskes wrote in message <35E6D862.2C05 at bbsrc.ac.uk>...
>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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