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Molecular Techniques and Methods

Isolation of Plasmid DNA from E. coli (Alkaline Lysis Method)

Copy Right 2001/ Institute of Molecular Development LLC

INTRODUCTION

MATERIALS AND SOLUTIONS 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 8,500 rpm for 3 min. 2. Remove supernatant. Completely 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 several times.

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4. Incubate tubes at room temperature for 5 minutes. 5. Add 9 ml of Solution-3. Mix well by inverting tubes several times. Cell debris containing chromosomal DNA, protein, and lipids, etc, should be visible. 6. To precipitate cell debris, centrifuge tubes at 10,000 rpm for 5 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 3,000 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 500 ul of TE buffer (pH8.0) by vortexing completely. 12. Transfer DNA solution in micorfuge tubes. 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 a new microfuge tube. 19. Add 30 ul of RNase A (DNase-free) (10 ug/ul) and incubate at 37oC for 30 min. 20. Add 60 ul of 3 M NaOAcpH5.2 and 500 ul of Phenol:CHCl3:IAA for extraction. 21. Vortex vigorously and centrifuge at 12,000 rpm for 5 min. 22. Transfer upper layer to a new microfuge tube. 23. Add 500 ul of CHCl3:IAA for extraction. 24. Vortex vigorously and centrifuge at 12,000 rpm for 5 min.

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25. Transfer upper layer to a new microfuge tube. 26. Add 500 ul iso-Propanol and vortex briefly. 27. DNA thread should be formed without centrifugation. Let precipitate and Using 200 ul pipette tip, carefully take out DNA thread to a new microfuge tube. Do not centrifuge in this step. Centrifugation precipitates tRNA toegther. 28. Wash DNA pellet by adding 1 ml of 70% ethanol. 29. Vortex vigorously and centrifuge briefly. 30. Remove ethanol and wash with 70% Et-OH two more times to remove any contaminating tRNAs. 31. Remove ethanol completely and air dry for 5 min. 32.Resuspend DNA pellet in 400 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. 33. Continue purification by loading sample into silica resin column. See Regeneration of Silica Based Resin (Ex. Qiagen Plasmid Purification Resin).

NOTES

KIT INFORMATION

REFERENCES

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