Need a rapid method to check transgenicity in mice? in mice

Tom Vasicek 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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