Best protocol to prepare BAC DNA
BL8KIE at WHY.NOT
Sat Sep 12 05:40:22 EST 1998
On Fri, 28 Aug 1998 17:18:42 +0100, clive tregaskes
<clive.tregaskes at bbsrc.ac.uk> wrote:
>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?
>> 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.
>Institute for Animal Health
>clive.tregaskes at bbsrc.ac.uk
>The longer I work in science the more I believe in magic
Wussup? I done hundreds of these preps with 500 ml BAC grown o/n in
TB. Yer wasting time and resources and getting an inferior product
messin' round with KITS that're made for teeny weenie lil' plasmids.
Get back to basics and prep 'em the old fashioned way.... by hand!
Some tips to keep in mind are... there's gonna be a LOT of crap in the
tubes and so extra spinnage and transferance of the super is
recommended. Also a blessing is the wonderous siliconized glass wool,
use this as a strainer after soln. III. And remember these are HUGE
inserts and sensitive to shearing so put that mechanical vortexer to
the side, when mixing soln. II and III in there (at this point I'd
have 'em in 50 mL falcon tubes) just *snap* you're wrist sharply a
couple to three times to insure mixture. spin the puppy in a bucket
at 4000 or so then go to yer fixed rotor beckman and crank it.
*STRAIN HERE* After that transfer to fresh 50 mL falco's and spin in
bucket again at 4000. This should leave you with spanking clean DNA.
after that it's just RNASE and whatever additional cleaning you wanna
500 mL culture
spin in the big thingie
throw 30 mL clean wawa and put in falcons.
spin at a reasonably gentle speed like 3k
toss the wawa
add solns 1, 2 and yeah 3.
spin as per that gibberish I wrote above
split into 2 tubes and precipertate with Iso propanol at RT overnight,
this keeps the additional crap from coming over as much.
ps or cc to your hearts content.
There's a bucha mod's you can make to it but this it the BEST for high
volume, like doin 30+ a day.
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