Alkaline Lysis - plasmid isolation
Alkaline lysis is the method of choice for isolating circular plasmid DNA, or even RNA, from bacterial cells. Alkaline lysis depends on a unique property of plasmid DNA: it is able to rapidly anneal following denaturation. This is what allows the plasmid DNA to be separated from the bacterial chromosome.


Procedure

1.         Spin 1.5 ml of fresh overnight culture cells for 5 minutes at 20,000 x g.

2.         Discard the supernatant.

3.         Add 100 µl of buffer TEG and resuspend your cells.

4.         Add 200 µl of 0.2 M NaOH, 1% SDS and mix GENTLY by inversion 3 times. Leave on ice for 5 minutes.

5.         Add 150 µl of cold 5 M KOAc and mix GENTLY by inversion 3 times. Leave on ice for 5 minutes.

6.         Add 2 volumes of EtOH. After 5 minutes, spin for 5 minutes at 12,000 rpm.

7.         Remove and discard supernatant. Add 500 µl of 70% EtOH to pellet and vortex briefly. Spin for 2 minutes at 12,000 rpm.

8.         Remove and discard the supernatant and allow the pellet to dry at room temperature, with the tube open upside-down on a paper towel. Resuspend in 50 µl of TE (Tris-EDTA Buffer).

Materials necessary
TEG Buffer
Fresh overnight cultures
0.2 M NaOH
1% SDS
5M Potassium acetate (KOAc)
100% Ethanol
70% Ethanol
TE

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