DNA Extraction from ES cells in 24 well plates
Note: Some enzymes, such as Xho I, do not cut ES cell DNA due to CpG island
methylation. Check the latest version of the New England Biolabs catalogue to
determine whether your enzymes will cut or not.
Lysis Buffer:
20 mM Tris HCl pH 8.0
10 mM EDTA
0.5% SDS
100 mM NaCl
100 ug/ml Proteinase K (5 ul 20 mg/ml stock per 1 ml buffer)
Stock protease K, freeze -20oC. Add fresh each use.
1. When cells in well are very confluent and the media is very orange/yellow, it is time to
lyse. The cells do not look "good" at this stage, but will give DNA.
2. Wash cells 1X with 1X PBS. (Only if cells are well stuck to bottom of plate.) Can
omit. Also can pour off media and freeze plate in-20 until ready to continue.
3. Add 0.3 ml lysis buffer containing proteinase K to cells in wells.
4. Lyse either in the plates, or transfer cells in lysis buffer to eppendorf tubes if you want
to lyse some wells, but not all wells in a plate. The cell/lysis buffer solution is very
goopy and viscous. All the contents will transfer together as a glob. This can be
accomplished with 1000 ul tips, but there will be strings of the material that you have to
"cut off" from the tip.
5. Cover plate with parafilm and incubate 1 hour at 55oC (preferably with shaking). The
temperature and time requirements of this step appear to be very flexible. You can lyse at
37oC indefinitely, and you can store lysed cells at room temp or 4oC. The only critical
thing is to not let the plates dry out.
6. Transfer lysate with wide bore pipette into Eppendorf tube. 55oC overnight.
7.Add 300 ul isopropanol to each tube (lysed volume) and invert a few times. (You
should be able to see a very thin strand of DNA). Centrifuge and discard supernatants.
8. Carefully wash pellet with 70% ethanol. Centrifuge and discard supernatants. (Be
careful: pellet is very slippery in 70% ethanol.)
9. Dry pellet completely but do not overdry. Resuspend in 40 ul 1X TE with RNase (10
ug/ml) 4oC overnight.
10. Store at 4oC
One hopes to get about 15 ug of DNA per well, but this may vary greatly.