Salting Out DNA prep. (Miller et al., NAR: 16, 1215, 1998)
1. Resuspend cell pellet in 18ml Lysis buffer, Shake
2. Add 1.2ml 10% SDS, Shake
3. Add 4.5ml Proteinase K, Shake
4. Add 24l RNase T1, Shake well
5. Incubate at 37oC overnight (or up to 3 days)
6. Add 6.6ml 6M saturated NaCl and shake vigorously for 3-4 min
7. Centrifuge at 22oC for 15 min at 2500 rpm
8. Transfer supernatant to clean polypropylene tube
9. Add 2 X the volume of 100% ethanol (roomtemp) shake gently to precipitate
DNA
10. Lift precipitated DNA with tip and transfer to 2ml TE in polypropylene tube
11. Incubate at 37oC for 2 hours
12. Put on Nutator at 4oC for 1 to 2 days to dissolve DNA
13. Determine concentration by reading OD260
Lysis Buffer: 10mM Tris-HCl, 400mM NaCl, 2mM EDTA
Proteinase K: 2mg/ml in 1% SDS, 2mM EDTA
RNase T1 (Roche #109193)
We routinely start with 5x107 cells, but the protocol can be scaled down
The 3-4 min shake at step 6 is very important for a good yield