REFERENCE: Molecular Cloning: A Laboratory Manual, 2nd ed., pp.1.25-1.28.
 

SOLUTIONS:

1. TE buffer 50 mM glucose, 25 mM Tris-Cl (pH 8.0), 10mM EDTA (pH 8.0); filter sterilized and stored at 4ºC.

2. 0.2N NaOH (freshly diluted from 10N stock), 1% SDS; This solution should be made up fresh on the day of use.

3. 3 M potassium and 5 M acetate. Store at 4ºC.

5 mM potassium acetate	60 ml
glacial acetic acid	11.5 ml
H2O	28.5 ml

 

METHOD:

1. Grow up a 5 ml culture overnight in the presence of the appropriate antibiotic.
2. Harvest 1.5 ml of culture by centrifugation in an Eppendorf tube.
3. Resuspend the pellet in 100 µl of ice-cold solution 1.
4. Store for 5 minutes at room temperature.
5. Add 200 µl of solution 2 and mix the contents by inverting the tube rapidly two or three times. Do not vortex.
6. Store for 5 minutes on ice.
7. Add 150 ul of ice-cold solution 3 and mix by vortexing in an inverted position.
8. Store for 5 minutes on ice.
9. Centrifuge for 5 minutes in a Eppendorf centrifuge at 4ºC.
10. Transfer supernatant to a fresh tube and add an equal volume of phenol/chloroform. Mix by vortexing the tube and centrifuge for 2 minutes in an eppendorf centrifuge at room temperature.
11. Transfer the top layer to a fresh tube and add two volumes of ethanol. Vortex gently and let stand at room temperature for 2 minutes.
12. Centrifuge for 5 minutes in an Eppendorf centrifuge at 4ºC.
13. Remove supernatant and add 1 ml of 70% ethanol. Vortex briefly and recentrifuge.
14. Remove supernatant and dry the pellet briefly in a vacuum dessicator.
15. Add 50 µl of TE (pH 8.0) containing DNase-free pancreatic RNase (20 µl/ml).