Genotyping from mouse tails

Chen-Ming Fan Lab,Carnegie Institute of Washington
(Laura’s method)

All pipets must be filter tipped
Be very careful to prevent cross contamination of samples
Change gloves often
Do not vortex the tubes
1. Place tail tip in 500 uL of Transgenic lysis buffer (may be stored at 4oC if
desired). Add 5 ul of 10 mg/mL Proteinase K. Set tubes in 55oC water bath
overnight. (may go up to 48hrs.)
2. Pellet debris by spinning in centrifuge for 2 min 13K rpm
3. Take off supernatant. Add 500uL of isopropanol. This will precipitate the DNA.
Invert the tube several times to mix.
4. Using a non filter pipet tip, spool out the DNA and place into a tube containing
500uL of 70% EtOH. This washes the DNA.
5. Take tip out with DNA still spooled around it. Tap on a kimwipe to wick off
excess ethanol (be careful to avoid cross-contamination here. Use a different spot
of the kimwipe each time to wick off the ethanol)
6. Place pipet tip with DNA in 200 uL of TE pH 7.5. This will dissolve the DNA.
Place tubes at 55oC for a while (1hr to overnight) to completely dissolve the DNA.
7. Use 1uL of the dissolved DNA for PCR analysis
Solutions:
Transgenic lysis Buffer: TE pH 7.5
For 100mLs For 100 mLs
10 ml 1M Tris pH 8.5 1ml 1M Tris pH 7.5
1mL 0.5 M EDTA 200 uL 0.5M EDTA
1mL 20% SDS
4 mL 5M NaCl