Alkaline Lysis Plasmid Miniprep

Wednesday, October 01, 2003

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

Procedure
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 祃 of ice-cold solution 1.
4. Store for 5 minutes at room temperature.
5. Add 200 祃 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 4篊.
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 4篊.
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 祃 of TE (pH 8.0) containing DNase-free pancreatic RNase (20 祃/ml).

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

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 4篊.
60 ml of 5 mM potassium acetate
11.5 ml of glacial acetic acid
28.5 ml of H2O
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Alkaline Lysis Plasmid Miniprep