Cfrench:maxipreps1

Plasmid DNA maxipreps from E. coli

Plasmid DNA maxiprep protocol for E. coli

• 1. Transfer 50 ml sterile LB aseptically into a 250 ml flask and add the appropriate antibiotic (eg, 50 microlitres of 100 mg/ml ampicillin). Inoclulate from a plate and incubate at 37˚C overnight with shaking.

• 2. Transfer the culture to a 50 ml Falcon tube. Spin at about 3500 to 4000 rpm for 10 minutes in the Centaur centrifuge with a properly balanced counterweight tube.

• 3. Pour the supernatant back into the flask and put it out to be autoclaved.

• 4. Resuspend the cells in 5.4 ml water. Add 15 microlitres of RNAse A stock solution (5 mg/ml in 50 mM EDTA, pH 8).

• 5. Add 3.0 ml of 0.4 M NaOH [CAUTION: NaOH is caustic! Wear gloves and eye protection!] and 0.6 ml of 10% w/v SDS. Mix by swirling. The solution should become clear and viscous.

• 6. Add 4. 5 ml of cold solution 3 (stored in the refrigerator) and mix by swirling. A precipitate should form.

• 7. Incubate on ice for 10 minutes.

• 8. Spin in the Centaur centrifuge for 10 minutes with a properly balanced counterweight tube.

• 9. Carefully transfer the supernatant to a fresh 50 ml Falcon tube and discard the pellet.

• 10. Add 28 ml of 100% ethanol and mix by swirling.

• 11. Incubate in the freezer for 30 minutes.

• 12. Spin in the Centaur centrifuge for 10 minutes with a properly balanced counterweight tube.

• 13. Pour off the supernatant and discard it.

• 14. Wash the pellet in 10 ml of 70% ethanol. Spin again briefly if necessary to get the pellet back to the bottom of the tube. Pour off and discard the 70% ethanol.

• 15. Dissolve the pellet in approximately 1 ml of EB. Transfer to a 1.5 ml microcentrifuge tube for storage.

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