Methods for Mouse Genotyping by PCR (protocol 2)
Transgenic Genotyping from Tail Biopsies
Harvard University—MCB Department
/ HSCI
- Remove .5-1 cm of the tail and place in 1.5 ml Eppendorf tube. (Store at -20oC until ready to digest).
- Digest in Lysis Buffer*
+ Proteinase K (to 200 ug/ml final conc.).
- Incubate in 55oC water bath overnight. (Vortex 1x after 1-2 h).
- Add .5 ml Phenol:Chloroform:Isoamyl
alcohol (25:24:1) to each tube and vortex for 30 sec.
- Spin at top speed in a microcentrifuge for 5 minutes.
- Transfer upper (aqueous)
phase to new tube; make sure no debris from the interface is transferred.
- Add 1 ml of 100% EtOH.
- Vortex briefly or shake. Stringy
white precipitate (the genomic DNA) should now be visible.
- Spin briefly (<1 min) just enough to get the DNA to cling to the plastic,
and decant supernatant.
- Wash with 1 ml of 70% EtOH.
- Let air dry until the pellet becomes partially translucent, but do NOT over-dry,
or the DNA will not go into solution any longer.
- Redissolve the pellet in 100 ml TE, pH 8.0.
- Check concentration, and calculate the
total yield, which should be around 10 to 50 mg.
- Use 100 ng for subsequent PCR analysis.
*Lysis Buffer:
- 10 mM Tris-HCl, pH
8.0
- 25 mM EDTA, pH 8.0
- 100 mM NaCl
- 0.5% SDS