Conklin Lab
Tail Tip Digest Protocol
(Modified protocols
from Hanahan Laboratory at UCSF)
- Cut 1/16"
to 1/8" of mouse tail from mouse using a razor blade.
- Place tails
in labeled 1.5 ml eppendorf tubes.
- Prepare digest
mixture:
Add 17 microliters tail tip buffer and 3 microliters proteinase K (20 mg/ml)
for a total of 20 microliters
- Add 20 microliters
of digest mixture into tubes containing tail tip.
- Incubate tubes
in a 55 deg C water bath for 1-2 hours.
- Remove tubes
from water bath. Spin briefly for 10 seconds.
- Add 500 ul of
milli-Q water to each tube.
- Boil samples
for 5 minutes. Place weight on top of tubes to prevent caps from popping.
- 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.
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