One step 'miniprep' method for the isolation of plasmid DNA protocol
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Solutions/reagents:
- overnight E.coli culture
- <a name="phenol : chloroform : isoamyl alcohol(25:24:1)">phenol : chloroform : isoamyl alcohol(25:24:1)
<tab>(phenol saturated with TE(10mM Tris, 7.5, 1mM EDTA) prior to mixing with chloroform and isoamylalcohol)</a> - isopropanol
- 70% ethanol
- 100 µl/ml RNAse
Equipment:
- Centrifuge
- Sterile 1.5-ml microcentrifuge tubes
Steps:
- Measure out 0.5 ml of overnight E.coli culture into sterile 1.5-ml microcentrifuge tube (1).
We routinely grow our cells in 'standard 1' bacteriological media supplied by Merck, Germany. - Measure out 0.5 ml of <a href="#phenol : chloroform : isoamyl alcohol(25:24:1)" >phenol : chloroform : isoamyl alcohol(25:24:1)</a> into sterile 1.5-ml microcentrifuge tube (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.
- Meanwhile:
Set aside a fresh sterile 1.5-ml microcentrifuge tube (2). Call it Tube I.
Measure out 0.5 ml of isopropanol into Tube I. - Add 0.45 ml of top aqueous phase obtained after centrifugation.
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.
Discard solution. - Repeat Step 7.
Add 100 µl/ml RNAse to solution.
Resuspend pellet by vortexing/by shaking vigorously.
About 5-10ul of this DNA can now be cleaved with appropriate restriction enzyme(s) for analysis.
TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 11 mins
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