DNA sterilization before transfection

Richard Moore rmoore at doppel.ucsf.edu
Mon Aug 2 12:58:24 EST 1999


Dear bewildered

I'm not sure of your cell type and whether you are doing stable or
transient transfections, nor am I sure of your protocol but is sounds as
if you are making Qiagen quality DNA (dissolved in H20) then
precipitating it **again** in ETOH. Does your DNA really need to be this
clean? If not, I would recommend that you dispense with this last
precipitation step. (Unless you are doing pronuclear injection for Tg
mice). I guess you are using EtOH because people say that isopropanol
-derived DNA is not as clean as DNA made by precipitation by ETOH. Maybe
so, but after Qiagen treatment that DNA is fairly clean anyway.  I'd say
that you are making DNA that is cleaner than you really need for most
applications. IMHO  I'd also dispense with the tissue culture hood and
invest in some aerosol free tips and just wipe down your Gilsons in 70%
EtOH before making your DNA.  I only ever used the hood for the actual
transfection and I do not routinely use antibiotics in my ES cell
medium.

This is what I do to make targeting vector quality DNA:

Grow E.coli in 50-100ml with 100ug/ml Amp for 18hrs. (Avoid upper end of
acceptable culture vol/qiagen column size)
Spin the little devils down and follow the Qiagen protocol for 500ml
tip. (seems like overkill but you won't overload the column with protein
etc this way)
Isopropanol precipitation at room temp, resuspend pellet in 1ml 70%EtOh
and transfer to 1.5ml Eppendorf.
Spin pellet down, discard 70%ETOH, wash again. Remove 70% EtOH with your
pipette (use p20 to get last few ul) and dissolve in H20.
I don't leave the DNA out to dry it...because i)  I don't have the
patience to wait, ii) sods law says that some wee bug will float into my
tube and iii) the DNA will be more difficult to dissolve.

This should be clean enough for transfection. I usually get 200ug+ of
15Kb Bluescript based targeting vector.

YOu don't need this for transient transfections but for stable
transfections I linearize the DNA with a single cutter then remove the
enzyme using small Qiagen spin columns (each has a 10ug max DNA binding
capacity).

I hope this helps.

--
Richard C Moore
Prusiner Lab
University of California San Francisco
Tel: (415) 502 1949
Fax: (415) 476 8386

rmoore at itsa.ucsf.edu
-------------- next part --------------
An HTML attachment was scrubbed...
URL: http://iubio.bio.indiana.edu/bionet/mm/methods/attachments/19990802/ca81fe1b/attachment.html


More information about the Methods mailing list