Smash and Bash DNA Isolation
1. Grow strain to saturation with selection for the plasmid. Harvest
1.5mLs of O/N culture in microfuge tube (with screw-on tops).
Resuspend in 0.2mLs TSENT. (Can freeze pellets O/N if necessary).
2. Add 0.3g (about 150uL worth) of glass beads to the resuspended cells.
Then add 0.2mLs of phenol/chloroform/IAA. Vortex 2 min.
3. Spin 5 min (full speed, 16K RPM). Transfer aqueous phase to a new
tube (normal eppendorf).
4. Ethanol ppt 1 time:
a. Add 10uL 3M NaOAc pH 5.2 and 500uL 100% EtOH and vortex.
b. Spin 5 min at 16K RPM and remove supernatant.
c. Add 500uL cold 70% EtOH.
d. Spin 5 min at 16K RPM and remove supernatant.
e. Spin again briefly and remove residual liquid with a pipet
f. Let air dry 5-15 min.
5. Resuspend in 50uL TE or ddH2O.
TSENT
2% Triton X-100
1% SDS
1 mM EDTA
100mM Na Cl
10mM Tris-HCl, pH. 8.0
100mLs
10mLs, 20%
5mLs, 20%
0.2mLs, 0.5M
2mLs, 5M
1mL, 1M