Gabriel Kaufman November 25, 2010Fritz lab

GenotypingDigestion and DNA extraction

Digestion1. Label 1.5-mL microcentrifuge tubes with mouse ID number.2. Obtain ear punch/tail from mouse of interest and transfer to labeled 1.5-mL

tube(Take ear punch from live mice/tails from dead mice. Clean ear punch/scissors in between mice with 70% ethanol to avoid tissue cross-contamination).

3. Place on ice until digestion. Samples can be stored at -20°C before digestion.4. Add 100 µL/ear punch / 500 µL/tail of digestion buffer with proteinase K (see

below for recipe) per tube to fully cover the tissue – quick-spin to ensure that the tissue is in the buffer.

5. Incubate for approximately 12 hours (overnight) at 55°C in heat block with occasional vortexing.

6. Vortex tubes for 10 seconds to complete tissue dissociation.

Digestion buffer(can be prepared without proteinase K and stored at room temperature)

50 mM Tris-HCl pH 8.0100 mM NaCl2.5 mM EDTA pH 8.00.5% (v/v) SDS

Add immediately before use:1 mg/mL proteinase K (stock: 10 mg/mL in ddH2O, stored at -20°C)

DNA extraction1. Centrifuge tube(s) at 12 000 x g for 10 minutes.2. Transfer 70/300 L (ear/tail) of the intermediate phase (avoid the debris on

top and the pellet!) into a fresh (labeled!) tube. 2 µL of this crude digest can sometimes be used directly for PCR, but must be validated for each genotyping protocol.

3. Add 300 µL isopropanol per tube to precipitate the DNA. Incubate 10 minutes at room temperature.

4. Centrifuge tubes at 12 000 x g for 10 minutes. Remove isopropanol supernatant by decanting and delicately blotting tubes on paper towels.

5. Add 500 µL of 75% ethanol to wash the DNA.6. Vortex tubes briefly and centrifuge at 7 500 x g for 5 minutes.7. Remove ethanol supernatant completely – use 0.5-10-µL micropipette tips and

P10 micropipettor to ensure complete removal (but do NOT aspirate the DNA pellet).

8. Dry tube in heat-block at 37°C until no more drops are visible (usually 2-3 minutes).

9. Air-dry on bench for 5 minutes – do NOT over-dry, or else the DNA will become difficult to resuspend.

10.Resuspend the DNA in 20 µL autoclaved ddH2O: GENTLY pass through pipette tip a few times in order to aid resuspension. DO NOT pipette vigorously as this will shear the DNA.

11. Incubate for 15 minutes at 65°C in heat block to complete resuspension of DNA.

12.Measure DNA concentration by NanoDrop spectrophotometry.13.Store extracted DNA at -20°C for at least three hours.

Use 500 ng DNA per reaction for PCR genotyping

Gabriel Kaufman November 25, 2010Fritz lab

OR

Use DNA at three dilutions for PCR genotyping –1:1 (2 µL in the PCR)1:10 (1 µL into 9 µL ddH2O; use 2 µL of this diluted mixture in the PCR)1:20 (1 µL into 19 µL ddH2O; use 2 µL of this diluted mixture in the PCR)