Lab Protocols‎ > ‎Protocols/Methods‎ > ‎

Tail Digest

*all except sFRP5 mice*

10x Modified Gitschier Buffer (10x MGB)
223 mL        Tris (1.5 M, pH 8.8)= 670 mM Tris
10.97 g        Ammonium Sulfate =166 mM Ammonium Sulfate
3 g               Magnesium Chloride= 65 mM Magnesium Chloride
Fill to 500 mL with Milli-Q H2O, mix well and autoclave

1x MGB (40 mL)
354. mL        Milli-Q H2O
4 mL             10x MGB
400 μL          2- Mercaptoethanol (final 1%)
200 μL          Triton X-100 (final 0.5%)

Pro K Mastermix (Per Sample)
1 mL             1x MGB
50 μL            Pro K solution (20 mg/mL)
[Ex: for 15 samples, 1500 μL 1x MGB + 75 μL Pro K Solution]

METHOD:
1. Clip 2-5 mm mouse tail with a clean razor. Place in labeled 1.5 mL tube and store at -20 ̊C until further processing.
2. Add 100 μL 1x MGB and heat at 95 ̊ C for 10 minutes.
3. Allow to cool for 5-10 minutes to about 55-65 ̊ C. Vortex and centrifuge
4. Add 100 μL Pro K mastermix. Vortex and spin.
5. Incubate at 55 ̊ C for 30 minutes. *See step 8*
6. Vortex on high for one second.
7. Repeat steps 5 and 6 four or five times.
8. Step 5 may be extended to an overnight digest. If this is done, ignore step 7.
9. When tail is fully digested, vortex and the pulse the tubes in the centrifuge.
10. Heat at 95 ̊C for 10 minutes to deactivate the Pro K.
11. Centrifuge at maximum speed for 5 minutes. Store the digested tail DNA at -20 ̊C.
12. Use 1 μL of tail prep for 50 μL PCR reaction.