Extraction of Plasmid DNA from bacteria (Alkaline lysis method)

Principle:

The bacterial cells are lysed with lysozyme and SDS at high pH and the lysate is then neutralized. The plasmid undergoes renaturation but not the chromosomal DNA. The later gets precipitated out in the form of protein-DNA-SDS complex. Subsequent deproteinization with phenol: chloroform, plasmid DNA is precipitated with ethanol by spinning at high speed. 

Requirements:

1. Overnight Log- phase culture in LB broth (10 g tryptone, 5 g yeast extract and 10 g NaCl, 1L DW)
2. Solution I (Lysis buffer): 25 mM Tris, 50 mM Glucose, Lysozyme (GNB: 0.5 mg/ml, GPB: 3-5 mg/ml), pH 8.0
3. Solution II (Lysis): 0.2M NaOH, 1% SDS 
4. Solution III: 3 M Sodium acetate, pH 4.8 by acetic acid
5. Phenol: Chloroform (1:1)
6. RNase A: (1 mg/ml in 5 mM Tris-HC1, pH 8.0)
7. Absolute ethanol/Isopropanol
8. 70% ethanol
9. TE buffer: 10 mM Tris, 1 mM EDTA, pH 8.0; Autoclave before use
10. Water bath
11. Cooling centrifuge (upto 20000 rpm)
12. Micropipettes (1 – 10, 10 -100, 100 – 1000 µl ) and sterile tips

 Procedure: 

1. Take 1.5 ml of overnight LB-broth culture of bacteria in a MFT and spin at 5000 rpm for 2 min.
2. Remove the supernatant and spin once with same volume as above to collect more cell mass.
3. Remove the supernatant.
4. Add 100 µl Sol. I. Re-suspend and keep for 30 min at RT.
5. Add 200 µl freshly prepared Sol. II and mix gently (Do not vortex). Keep for 5 min at 4°C.
6. Add 150 µl ice cold Sol. III and mix gently. Keep in ice bath for 10 min.
7. Spin at 10000 rpm for 5 min at 4°C. Collect Sup-T into new MFT.
8. Add 1-2 µl of RNase and incubate for 10 min at RT.
9. Add equal vol. of Phenol:Chloroform (1:1), mix gently and keep for 10 min at RT.
10. Centrifuge at 8000 rpm for 10 min at 4ºC and collect the supernatant in a new sterile MFT.
11. Add two vol. of absolute ethanol and stand it for 10 min in cold.
12. Spin at 13000 rpm at 4ºC for 10 min.
13. Remove the Sup-T into new MFT and add 1000 µl of 70% ethanol. Mix well and keep at 4°C for 30 min.
14. Spin at 13000 rpm at 4ºC for 10 min. Remove Sup-T.
15. Dissolve the pellet collected during spin in 50 µl TE and store in deep freeze.