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dtmc

Houston, Texas

BCM is a research-intensive medical center dedicated to a rapid pace of discovery and its equally rapid translation to patient care.
Mouse Embryonic Stem Cell Core Facility
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Mini Southern Analysis

Materials:

  • Lysis Buffer:

    10mM Tris, pH 7.5
    10mM EDTA, pH 8.0
    10mM NaCl
    0.5% Sarcosyl
    2mg/ml Proteinase K (added fresh)
  • 75mM NaCl in EtOH; prepared fresh
  • 70% EtOH
  • Restriction Enzyme Cocktail

Procedure:

  1. Wash plates twice with PBS using 100ul/well.
  2. Place 50ul of Lysis Buffer as prepared above.
  3. Incubate overnight at 55-60°C in a humidified chamber.
  4. Prepare a fresh solution of 75mM NaCl in EtOH (add 150ul of 5M NaCl per 10 mls of cold, absolute EtOH and mix well. The salt will precipitate but this is of little consequence.
  5. Using a multichannel pipette, add 100ul of NaCl/EtOH solution per well. Allow the plate to incubate on the bench top at room temperature for 15 to 30 minutes or until the precipitated DNA is clearly visible under low power magnification. The DNA adheres to the plastic, around the perimeter of each well.
  6. Invert plate and discard the solution. DNA should remain in the plate.
  7. Add 70% EtOH to each well in order to wash and invert plate to discard. Repeat this step 2-3 times.
  8. After final wash, allow the plate to dry at room temperature for 10 minutes.
  9. Prepare the restriction enzyme cocktail and place 40ul of restriction enzyme cocktail (1X restriction buffer for enzyme used; 1mM Spermidine; 100ug/ml BSA; 10-15 units of enzyme) per well using a multichannel pipette. Pipette up and down in each well to mix the solution with the contents of the well. Use a clean tip for each well.
  10. Incubate plates overnight at 37°C in a humidified chamber.
  11. Run samples on agarose gel next morning.
  12. After electrophoresis is complete, transfer the DNA to a membrane for blotting.