Total DNA Preperation for Dicty

Procedure

1. Collect approx. 5x10^8 cells and wash twice with cold, sterile TE. (Spin in table-top centrifuge 3-5min)
2. Resuspend in ice-cold TE to 6ml in a 15ml polypropylene tube.
3. Add 0.5ml 30%Sarkosyl (N-Lauroylsarcosine, Na) and mix by inverting several times. (go to next step immediately!)
4. Phenol extract on rotating wheel approx. 10 min: Add equal volume of phenol. After shaking, spin in clinical 'fuge at 3k rpm for 40min. Remove sup into clean tube using wide-bore pastuer pipettes. (If yields are poor extract interface w/ 1-1.5ml TE)
5. Phenol-chloroform extract on rotating wheel approx 10 min. (Spin the following steps for 10-15 minutes only)
6. Add 50ul of a 10mg/ml RNAse stock. Incubate 37C for 1hr.
7. Add 50ul of a 5mg/ml proteinase K stock. Incubate 37C for 1hr.
8. Extract w/ phenol, phenol-chloroform, and chloroform again.
9. Ethanol precipitate: add 1/3 volume 7.5M ammonium acetate and then two volumes (total volume) of ethanol. Rinse ppt. w/ 70% Ethanol. Bring up in TE and repeat ethanol precipiation.
10. Resuspend in 3ml TE (This may take some time). Carefully vortex.
11. Read O.D. 260/280
12. Run sample on gel w/ standards to quantify DNA.