Plasmid?DNA?Isolation?from?Bacteria

Materials:

1.TENS solution:

10 mM Tris (pH to 7.5)

1 mM ethylenediaminetetraacetic acid (pH to 8.0 to dissolve)

0.1 N sodium hydroxide

0.5 % sodium dodecyl sulfate

2.3 M Sodium acetate, pH 5.2

3.Pre-chilled (at -20 ℃) 100 % ethanol

4.70 % Ethanol

5.Distilled water

6.Overnight bacterial culture (LAB 5)

Supplies:

1.Micropipetter and tips

2.Vortex mixer

3.Microcentrifuge and tubes

Procedures:

1.Spin 1.5 ml of overnight bacterial culture for 30-60 seconds in a microcentrifuge. (observation: bacteria form a pellet at the bottom of the tube.)

2.Decant supernatant, leaving 50-100 μl in the tube.

3.Vortex to resuspend the bacteria pellet completely.

4.Add 300 μl of TENS solution.

5.Vortex for 5 seconds to mix. (observation: the contents of the tube should become slimy.)

6.Add 150 μl of the sodium acetate.

7.Vortex for 5 seconds to mix.

8.Spin for 2 minutes in a microcentrifuge. (observation: a white pellet, containing bacterial debris, is formed at the bottom of the tube.)

9.Transfer supernatant to a fresh tube.

10.Add 0.9 ml of pre-chilled 100 % ethanol.

11.Spin for 5 minutes in a microcentrifuge. (observation: a white pellet, containing plasmid DNA and bacterial RNA, is formed at the bottom of the tube.)

12.Discard supernatant and add 1 ml of 70 % ethanol.

13.Discard the ethanol and add another 1 ml of 70 % ethanol.

14.Withdraw and discard as much liquid (ethanol) as possible then air-dry the pellet.

15.Resuspend the pellet in 30 μl of distilled water and keep at 4 ℃or -20 ℃.

Results:

1.Plasmid DNA is now ready for estimation of DNA concentration (LAB 4) followed by restriction digest (LAB 3).

2.Typical yield is 2-3 μg.