ES Cell DNA Preparation

ES cell lysis buffer (KT)

• 20 mM tris pH 8.0
• 10 mM EDTA
• 100 mM NaCl
• 0.5% SDS

Proteinase K

• stock at 20 mg/ml in H 2O
• aliquot and store at –20 oC
• Use at 1 mg/ml = 1:20 dilution = 50 ul/ml

TE

• 10mM tris pH 8.0
• 1mM EDTA

Procedure

• Wash cells (100 mm plate) with 5 ml PBS.
• Add 5 ml ES cell lysis buffer to plate.
• Add 250 ul Proteinase K to each tube. Parafilm plate.
• Incubate 55 o overnight.
• Scrape each plate into 15 ml conical. Ethanol scraper between each plate.
• Add 5 ml isopropanol to each tube. ( ≥ lysed cell volume)
• Invert a few times. Should see DNA precipitate.
• Centrifuge 2000xg for 10 minutes.
• Dump off supernatant being careful not to lose pellet (should see visible white pellet on bottom of tube).
• Add 2 ml 70% EtOH (high quality).
• Vortex until pellet comes off bottom of tube.
• Centrifuge 2000xg for 10 minutes.
• Carefully dump off ethanol.
• Blot tube on paper towel and allow to drain.
• Dry completely.
• Add 500ul TE with Rnase (10ug/ml final) to dissolve DNA. (best to let it dissolve overnight at 4 oC), (can put at 55 o for a while to help dissolve DNA).
• After DNA has dissolved transfer to microfuge tube.
• Store at 4 oC, or at –20 oC for long term storage.