Need a rapid method to check transgenicity in mice? in mice
tvasicek at watson.princeton.edu
Mon Jan 29 10:36:00 EST 1996
In article <1996Jan29.124016.3925 at news.huji.ac.il>, Mirta Grifman
<mirtag at leonardo.ls.huji.ac.il> wrote:
> In our lab the standard method is to tail the mice and extract DNA from
> the tail and then PCR with primers for the transgene. The protocol seems
> quite old-fashioned and takes too much time. Wouldn't it be possible to
> cut some hairs and PCR from them?
Quick DNA for PCR (tail, toe, ear punch, or embryo fragment)
1. Place tissue in a microfuge tube or microtiter well with watchmaker¹s
forceps. Be careful not to cross-contaminate samples. If you use PCR
tubes, you can do steps 4-5 in the thermocycler. Store tissue samples at
-20, or use immediately.
2. Centrifuge tissue to bottom of tube.
3. Add 40 ul of digestion buffer for an ear punch; 200 ul for a very
small piece of tail. Scale the volume to the size of the tissue sample:
~5 to 10 ul/mg of tissue.
4. Digest at 55 to 65 degrees for 2 to 24 h. Occasional shaking (vortex
or invert several times every 15 min) speeds the process.
5. Denature proteinase K at 95 degrees for 10 min.
6. Use a multichannel pippet to transfer 1-2 ul to a 20 ul PCR; store DNA
at -20 degrees without further purification.
Digestion Buffer: --for 50 ml:
50 mM KCl 2.5 ml 1M
10 mM Tris-Cl pH 8.3 0.25 ml 2 M Tris
2 mM MgCl2 0.1 ml 1M
0.45% NP40 0.225 ml 10%
0.45% Tween 20 0.225 ml 10%
46.7 ml H2O
Add Proteinase K to 100 ug/ml just before use.
Thomas J. Vasicek, Ph.D. tvasicek at watson.princeton.edu
Department of Molecular Biology FAX: 609/258-3345
Princeton University Phone: 609/258-2899
Princeton, New Jersey 08544
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