One step 'miniprep' method for the isolation of plasmid DNA protocol
- overnight E.coli culture
- phenol : chloroform : isoamyl alcohol(25:24:1) (phenol saturated with TE(10mM Tris, 7.5, 1mM EDTA) prior to mixing with chloroform and isoamylalcohol)
- 70% ethanol
- 100 µl/ml RNAse
- Flasks of appropriate volumes
- Sterile 1.5-ml microcentrifuge tubes
- Measure out 0.5 ml of overnight E.coli culture into a sterile 1.5-ml microcentrifuge tube.
We routinely grow our cells in 'standard 1' bacteriological media supplied by Merck, Germany.
- Add 0.5 ml of phenol : chloroform : isoamyl alcohol(25:24:1).
- Vortex the mixture for 1 min .
Vortex at maximum speed.
Alternatively, vortex for 10 seconds and then transfer to eppendorf mixer or an over-the-top rotator for 5 minutes.
- Centrifuge at a speed of 12000 Xg for 5 mins at room temperature.
Set aside a fresh a sterile 1.5-ml microcentrifuge tube. Call it Tube I.
Measure out 0.5 ml of isopropanol into Tube I.
- Measure out 0.45 ml of top aqueous phase obtained after centrifugation into Tube I.
Vortex the mixture for a few secs.
Centrifuge at a speed of 12000 Xg for 5 mins at room temperature, gently aspirate out the supernatant and discard it.
Addition of salt and cooling is unnecessary.
- Add 0.5 ml of 70% ethanol.
Add ethanol carefully to the side of the tube.
- Repeat Step 7.
Add 100 µl/ml RNAse to solution.
Resuspend the pellet by vortexing/by shaking vigorously.
About 5-10ul of this DNA can now be cleaved with appropriate restriction enzyme(s) for analysis.