Bacterial Artificial Chromosome (BAC) DNA purification for pronuclear injection:
There are at least 4 methods for producing transgenic mice using BAC DNA.
Our preferred method is to microinject Pulse Field Gel Electrophoresis (PFGE) – purified BAC
insert. PFGE is used because DNA 15-20kb or larger in size will migrate with the same mobility
regardless of its size during continuous field electrophoresis. Having an alternating gradient
voltage will give better resolution of these larger molecules. This procedure takes longer than
normal gel electrophoresis due to the size of the fragments being resolved and the fact that the
DNA does not move in a straight line through the gel.
However, we know that not many people have access to or experience with this kind of
equipment and therefore can not give us PFGE purified BAC DNA.
We recommend using the QIAGEN Large-Construct Kit (which is for the purification of up to 50
μg BAC, PAC, and P1 DNA or up to 200 μg cosmid DNA, free of genomic DNA)
Catalogue number 12462
Cost ~$558 for 10 preps.
Combined with the last steps of YAC DNA purification method as described here:
http://www.cnb.csic.es/~montoliu/prot.html - which is essentially dialysis of the BAC DNA prep
on a floating 0.05um Millipore Membrane against an excess of 50-100 times microinjection
buffer with polyamines for a couple of hours.
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At this point a 260/280 ratio will be relevant as the dialysis will have removed a lot of
the contaminants from the sample. On its own, the QIAGEN prep will not be clean
enough for microinjection and a 260/280 ratio will not be relevant as the sample will
have contaminant agents in it which will corrupt your spectral reading.
A few pointers:
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Do NOT ethanol precipitate, treat with phenol or freeze down, please store at 4C.
The prep should be crystal clear – if you can see stuff in solution we will not inject it, you
will have to do dialysis
260/280 ratio usually won't be meaningful and is pretty much useless with BAC DNA
preps unless you dialise first since the standard BAC DNA procedure (i.e. Qiagen
columns) usually produce a DNA prep with contaminating agents that corrupt your
spectral reading.
To properly measure DNA concentration of BAC DNA preps the best solution is using a
fluorometer. Spectrophotometers or related apparatus (i.e. nanodrop) are less efficient
since they read a number of other stuff in the usually dirty BAC DNA preps and
therefore the concentration is nearly always wrong. Fluorometer reads in a 90 degree
angle on glass cuvetters and upon reaction with a fluorofor which only reacts with
dsDNA, no matter what else is in solution.
An even better estimation can be obtained by comparing your prep with a previous prep
for which you know the correct concentration and you compare the EtBr intensities on a
standard gel.
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If you can't precipitate how do you concentrate your usually low amount BAC DNA
prep? Will you need to concentrate it? DNA could be dropped down to 0.1 ng/ul and still
be reasonably possible to get several transgenic lines. Or you can use Centricon
microtubes to spin and concentrate samples.
There are 2 websites we recommend visiting before you start to get a feel for what is involved
and any alternative methods that might suit you better – please contact us before you submit
DNA to us:
Thom Saunder's Transgenic-facility web page at the University of Michigan where you'll find a detailed
protocols & comments on the preparation of BAC DNA for microinjections:
http://www.med.umich.edu/tamc/BACDNA.html
Lluis Montoliu’s webpage at the National Centre of Biotechnology, Madrid
Which has detailed protocols at: http://www.cnb.uam.es/~montoliu/prot.html