Mouse Tail DNA Analysis Protocol

Overview

List of reagents is not yet complete

Procedure

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.

References

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