This protocol gives very clean plasmid preps for restriction digests and cloning. However, due to the alkaline lysis step, the DNA is often nicked and may not give exceptional sequence data.
Solutions
TENS
0.1 N NaOH 1 ml 10N NaOH
0.2% SDS 1 ml 20% SDS
10 mM Tris 7.5 1 ml 1M Tris 7.5
1 mM EDTA 200 μl 0.5 M EDTA
up to 100 ml with Q
store at room temperature
3 M NaOAc 5.2
24.6 g anhydrous sodium acetate
pH to 5.2 with acetic acid and bring up to 100 ml with Q
Procedure
• Inoculate a 2 ml overnight culture with a single colony.
• Pellet 1.5 ml of the overnight culture and aspirate all but 50 ml of the LB.
• Resuspend the cell pellet in the remaining LB and add 300 ml TENS.
• Vortex, and add 150 ml 3M NaOAc 5.2.
• Vortex again and spin for 2 minutes in the microfuge. Discard the pellet using a toothpick.
• Add 300 ml phenol/chloroform, vortex and spin for 5 minutes in the microfuge.
• Retrieve 200 ml of aqueous supernatant and add 600 ml EtOH. Spin for 10 minutes in the microfuge, wash and dry the pellet.
• Resuspend in 40 ml TE. 5 ml is generally sufficient for restriction digests (add RNaseA).
