Mouse Tail DNA Analysis Protocol
List of reagents is not yet complete
Conklin Lab Tail Tip Digest Protocol (Modified protocols from Hanahan Laboratory at UCSF)
- 1. Cut 1/16" to 1/8" of mouse tail from mouse using a razor blade.
- 2. Place tails in labeled 1.5 ml eppendorf tubes.
- 3. Prepare digest mixture:
Add 17 microliters tail tip buffer and 3 microliters proteinase K (20 mg/ml) for a total of 20 microliters
- 4. Add 20 microliters of digest mixture into tubes containing tail tip.
- 5. Incubate tubes in a 55 deg C water bath for 1-2 hours.
- 6. Remove tubes from water bath. Spin briefly for 10 seconds.
- 7. Add 500 ul of milli-Q water to each tube.
- 8. Boil samples for 5 minutes. Place weight on top of tubes to prevent caps from popping.
- 9. Spin samples briefly. Store at 4 deg C until PCR.
Tail tip buffer:
- 50 mM Tris, pH 8.0
- 20 mM NaCl
- 1 mM EDTA, pH 8.0
- 1% SDS
- (adjust to proper volume using milli-Q water)
Conklin Lab PCR Tail Tip Analysis Protocol
- 1. Label PCR tubes.
- 2. Add 2.0 ul of digested mouse tail tip to the appropriate PCR tube.
- 3. Prepare PCR reaction mixture:
- 39.8 microliters Milli-Q water
- 5.0 microliters 10x PCR buffer +Mg
- 1.0 microliter 10mM dNTP
- 1.0 microliter 5' primer (25uM)
- 1.0 microliter 3' primer (25uM)
- 0.2 microliter Taq (added last)
- Total = 48.0 microliters
(Taq and 10x PCR buffer +Mg are from Boehringer Mannheim)
- 4. Add 48.0 microliters of PCR reaction mixture to each PCR tube.
- 5. Place tubes in a thermal cycler using the following conditions:
*94 deg C for 3 min *94 deg C for 30 sec *60 deg C for 30 sec *72 deg C for 1 min *go to step 2, 34 times *4 deg C forever
- 6. Analyze PCR product on a 2% agarose gel.
Relevant papers and books
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