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Materials:
Bradley Lysis Buffer
Stock Solutions Final Conc for 50mL
1M Tris-HCl (pH 7.5) 10 mM 500 uL
0.5M EDTA 10 mM 1 mL
10% SDS 0.5 % 2.5 mL
5M NaCl 10 mM 100 uL
H2O 45.9 mL
ProteinaseK (20mg/mL)
:add just before use as 1mg/ml final concentration
EtOH/NaCl Mix
Stock Solutions
100% EtOH 39.4mL
5M NaCl 600uL
Total volume 40mL
Methods:
- Grow ES cells in a 96-well plate to be over-confluent.
- Quickly invert the plate over to dump media and remove excess liquid by blotting on paper towels.
- Wash cells 1x with 150 uL PBS, then dump PBS
- Add 50 ul of Bradley Lysis Buffer containing proteinase K.
- Replace lid and seal the plate with parafilm. Put the plate into a humidified chamber
- Incubate in the humidified chamber O/N @ 60 degree.
- Allow the plate to cool to RT
- Add 100 uL ice-cold EtOH/NaCl mix to precipitate DNA and mix well. Then incubate the plate about 30 minutes at RT
- Spin in 96-well plate holder centrifuge, 2000rpm 10 minutes
- Invert the plate to decant liquid. Blot the plate on paper towels to remove access liquid.
- Add 150uL cold 70% EtOH and spin 10minutes at 2000 rpm to rinse the pellet. Decant supernatant.
- Repeat washing step ( #11) and air-dry DNA pellet until there is no detectable EtOH smell (approx 10-15 minutes)
- Add 30 uL of warm TE pH 8.0
- Cover Plate with wax cover and incubate at 56 degree ~10 mins