(NOTE: the DNA from the following protocol is suitable for restriction mapping, but NOT for sequencing)
TE/RNase
TE 10 ml
RNAse: 200 ul (Roche 1119915)
TENS: 100 ml
100XTE: 1 ml
10% SDS: 5 ml
10 N NaOH: 1 ml
water: 93 ml
1. Spin down 1 ml of an overnight culture in 1.5 ml Eppendorf for 1-1.5 min.
2. Aspirate supernatant, and add 50 ul of TE/RNAse (or 50 ul of Qiagen buffer P1 with Rnase)
3. Resuspend pellet (vortex very well to make sure that the pellet is entirely resuspended!).
4. Add 200 ul TENS.
5. Add 100 ul 3 M NaOAc pH 5.2.
6. Add about 100 ul chloroform (this helps to flatten the pellet after centrifugation).
7. Mix by inversion, then spin at top speed for 2 min.
8. Transfer supernatant to new tube.
9. Add 1 ml 100% ethanol and vortex.
10. Spin at top speed for 3 min. and aspirate off supernatant.
11. Wash with 0.5 ml of 70% ethanol, and spin for 2 min at 4 C
12. Aspirate supernatant, and air dry for 5 min.
12. Resuspend in 50 ul of TE.
13. Proceed with restriction digestion
(This was originally for 1.5 ml culture, with 300mcl TENS and 150mcl NaOAc)
(If you need to sequence the DNA from this preparation, you should follow this with either phenol—phenol/Chloroform—chloroform extraction or use the Qiagen PCR clean-up column.
