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Gabriel Kaufman Fritz lab

November 25, 2010

Genotyping Digestion and DNA extraction Digestion 1. 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 -20C before digestion. 4. Add 100 L/ear punch / 500 L/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 55C 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 8.0 100 mM NaCl 2.5 mM EDTA pH 8.0 0.5% (v/v) SDS Add immediately before use: 1 mg/mL proteinase K (stock: 10 mg/mL in ddH2O, stored at -20C) DNA extraction 1. 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 L of this crude digest can sometimes be used directly for PCR, but must be validated for each genotyping protocol. 3. Add 300 L 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 L 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-L micropipette tips and P10 micropipettor to ensure complete removal (but do NOT aspirate the DNA pellet). 8. Dry tube in heat-block at 37C 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 L 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 65C in heat block to complete resuspension of DNA. 12. Measure DNA concentration by NanoDrop spectrophotometry. 13. Store extracted DNA at -20C for at least three hours. Use 500 ng DNA per reaction for PCR genotyping OR Use DNA at three dilutions for PCR genotyping 1:1 (2 L in the PCR) 1:10 (1 L into 9 L ddH2O; use 2 L of this diluted mixture in the PCR) 1:20 (1 L into 19 L ddH2O; use 2 L of this diluted mixture in the PCR)