Best protocol to prepare BAC DNA
schneema at cmgm.Stanford.EDU
Thu Aug 27 11:51:49 EST 1998
QIAGEN has a modified procedure for BACprep - you should harass your
Qiagen-rep. to send it to you !
1) 500ml culture is too much - do 50-100mL and use midi-columns (ask
qiagen (in the BAC-proc.). with the 5oomL culture you'll run into trouble
with genomic DNA-contam. and overloading the column.
2) Apparently, the alkaline lysis step II is crucial in order not to get
contamination of your BAC-DNA with genomic Coli-DNA / that means 4-5min
only / on ice / mix gently / use fresh NaOH/SDS-sol. each time.
3) Elute the DNA from the column with 60 C warm H2O.
4) You won't get more than 5-20ug/prep. - use 5-10ug for a restr. digest
(use non-star activity ones = not EcoRI or BamHI / I prefer Xba I in
Pharmacia one-phor all buffer)
schneema at cmgm.stanford.edu
On 26 Aug 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?
> Jun-ichi Aikawa, Ph.D.
> University of California, San Diego
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