Mouse Tail DNA Analysis Protocol

From OpenWetWare

Revision as of 17:44, 24 June 2009 by Elana Urbansky (Talk | contribs)
(diff) ←Older revision | Current revision (diff) | Newer revision→ (diff)
Jump to: navigation, search

Contents

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

No further references are available at this time

Contact

  • Who has experience with this protocol?

or instead, discuss this protocol.


Personal tools