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.
