Solution-1 (500 ml) - Cell Resuspension Buffer
50 mM Tris-HCl (pH8.0) --------------------- 25 ml of 1 M Tris-HCl
10 mM EDTA -------------------------------- 10 ml of 0.5 M EDTA
10 mg RNase A (heat-treated) ---------------- 5 ml of RNase A (10 mg/ml)
Deionized H2O ------------------------------- 460 ml
Keep at 4oC.
Solution-2 (500 ml) - Cell Lysis Buffer
200 mM NaOH ------------------------------- 20 ml of 5 M NaOH
1% SDS -------------------------------------- 50 ml of 10% SDS
Deionized H2O -------------------------------- 430 ml
Solution-3 (3 M KoAc) (500 ml) - Neutralization Buffer
5 M Potassium acetate (pH4.8) ------------------ 245 g
Glacial acetic acid -------------------------------- 115 ml
Add deionized H2O to make a final volume of --- 500 ml
CRITICAL-Adjust pH to 4.8 with glacial acetic acid.
PROCEDURES
1. Pellet 150-200 ml overnight culture of E. coli by centrifugation at 12,000 rpm for 3 min.
2. Remove supernatant. Resuspend cells in 8 ml of Solution-1.
Important: Vortex to resuspend cells completely.
3. Add 9 ml of Solution-2.
Mix well by inverting tubes. Do not vortex.
4. Incubate tubes at room temperature for 5 minutes.
5. Add 9 ml of Solution-3.
Mix well by inverting tubes. Do not vortex.
Cell debris containing chromosomal DNA, protein, and lipids, etc, should be visible.
6. To precipitate cell debris, centrifuge tubes at 12,000 rpm for 10 minutes.
7. Transfer 25 ml supernatent to a new 50 ml disposable tube.
8. Add 25 ml of iPr-OH and mix well.
9. To pellet DNA, centrifuge tube at 4,000 rpm for 5 min.
Large pellet containing plasmid DNA, tRNA, and small RNA should be formed at the bottom of tube.
10. Remove supernatant completely.
11. Resuspend DNA pellet in 1 ml of TE buffer (pH8.0) by vortexing completely.
12. Divide and transfer DNA solution in micorfuge tubes (500 ul/tube) .
13. Add 500ul Phenol:CHCl3:IAA and vortex vigorously.
14. Centrifuge tubes at 12,000 rpm for 5 min.
15. Transfer upper layer to new microfuge tubes.
16. Add 500 ul CHCl3:IAA and vortex vigorously.
17. Centrifuge tubes at 12,000 rpm for 5 min.
18. Transfer upper layer to new microfuge tubes.
19. Add 30 ul of RNase A (DNase-free) (10 ug/ul) and incubate at 37oC for 1 hr.
20. Add 60 ul of 3 M NaOAcpH5.2 and 500 ul of Phenol:CHCl3:IAA for extraction.
Vortex vigorously and centrifuge at 12,000 rpm for 5 min.
21. Transfer upper layer to new microfuge tubes.
22. Add 500 ul of CHCl3:IAA for extraction.
Vortex vigorously and centrifuge at 12,000 rpm for 5 min.
23. Transfer upper layer to new microfuge tubes.
24. Add 500 ul Iso-propanol and vortex briefly.
25. DNA thread should be appeared. Using 200 ul pipette tip, carefully take out DNA thread to new microfuge tubes.
26. Wash DNA pellet by adding 1 ml of 70% ethanol.
27. Centrifuge briefly to pellet DNA at the bottom of tube.
28. Repeat steps 26, 27, two more times to remove any contaminating tRNAs.
29. Remove ethanol completely and air dry for 5 min.
30.Resuspend DNA pellet in 100-200 ul TE buffer.
DNA quality is good for transfection and sequencing.
It is not necessary to do column purification.
Optional: If Qiagene column is used for further purification, follow next step.
However, Qiagene column purification is not necessary.