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

Relevant papers and books

If this protocol has papers or books associated with it, list those references here. Below is an example for formatting purposes. See the OpenWetWare:Biblio page for more information.

1. Goldbeter A and Koshland DE Jr. An amplified sensitivity arising from covalent modification in biological systems. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6840-4. DOI:10.1073/pnas.78.11.6840 |
2. JACOB F and MONOD J. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol. 1961 Jun;3:318-56. DOI:10.1016/s0022-2836(61)80072-7 |
3. ISBN:0879697164

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