The wells should be at least ~80% confluent. These cells can overgrow since you are only trying to make a genomic prep.
1. Wash confluent cells in 12-well plates 1x with PBS
2. Add 400 ul of HMW buffer (see below) per well
3. Scrape cells with plastic scraper
4. Collect cells and transfer into 1.2 ml eppendorf tubes
5. Add 8 ul of 10% SDS and 25-30 ul of 10mg/ml of proteinase K
6. Incubate in a 55C waterbath overnight.
7. Next day add 400 ul of phenol and rotate at room temp. for 30 min.
8. Centrifuge at 14,000 rpms for 5 min at room temp
9. Transfer supernatant into new eppendorf tubes with 400 ul of phenol/chloroform
10. Rotate for 30 min. at room temp and centrifuge
11. Transfer the supernatant into new tubes, add 1-ml ethanol. Mix, genomic DNA should be visible.
12. Centrifuge at 14,000 rpms at room temp. for 5 min. Decant the supernatant.
13. Wash the pellet with 1 ml of 70 % ethanol 2X.
14. Dry pellet, (use speed vac for 8 min., or vacuum)
15. Add 100 ul of water and store at 4C overnight to dissolve completely.
HMW buffer
10mM Tris-HCl pH 8
150 mM NaCl
10 mM EDTA pH 8
autoclave, add 10% SDS to a 0.5% final concentration before use
NOTE: The DNA preparation is still crude and includes some RNA, so calculations using the spec. are inflated. The DNA conc. is still high, use ~ 10 ug for Southern blotting.
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