Molecular Techniques and Methods

Isolation of Plasmid DNA
from Large Volume of E. coli Culture
(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 ------------------------------ 147 g
    Glacial acetic acid -------------------------------- 115 ml
    Add deionized H2O to make a final volume of --- 500 ml
  • CRITICAL-Adjust pH to 4.8 with conc. HCl.




    PROCEDURES

    1. Pellet 150 ml overnight culture (16~18 h grown) of E. coli by centrifugation at 9,000 rpm.
  • To increase the plasmid yield, after 12-14 h incubation, add chloramphenicol and incubate additional 4 h before harvest.
  • As it reaches 9,000 rpm, stop centrifugation.

    2. Remove supernatant. Completely resuspend cells in 8 ml of Solution-1. Transfer to the 50 ml blue cap tube.
  • Important: Vortex to resuspend cells completely.

    3. Add 9 ml of Solution-2.
  • Mix well by inverting tubes several times.

    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 3,000 rpm for 5 minutes.

    7. Transfer supernatent (~25 ml) to a new 50 ml disposable tube.

    8. Add 25 ml of iPr-OH and mix well.

    9. To precipitate DNA, centrifuge tube at 3,000 rpm for 5 min.
  • Large pellet containing plasmid DNA and RNA should be formed at the bottom of tube.

    10. Remove iPr-OH completely.

    11. Resuspend DNA pellet in 500 ul of H2O by vortexing.

    12. Transfer DNA solution in a micorfuge tube.

    13. Add 500ul Phenol:CHCl3:IAA and vortex vigorously.

    14. Centrifuge tubes at 12,000 rpm for 3-5 min.

    15. Transfer upper layer to a new microfuge tube.

    16. Add 500 ul CHCl3:IAA and vortex vigorously.

    17. Centrifuge tubes at 12,000 rpm for 3-5 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 20-30 min.

    20. Add 70 ul of 3 M NaOAcpH5.2 and 500 ul of Phenol:CHCl3:IAA.

    21. Vortex vigorously and centrifuge at 12,000 rpm for 3-5 min.

    22. Transfer upper layer to a new microfuge tube.

    23. Add 500 ul of CHCl3:IAA.

    24. Vortex vigorously and centrifuge at 12,000 rpm for 3-5 min.

    25. Transfer upper layer to a new microfuge tube.

    26. Add 500 ul iso-Propanol and mix by inverting tube 3~5 times.
  • Do not vortex. tRNA may co-precipitate with plasmid DNA by vortexing.
  • DNA thread should be formed without centrifugation.

    27. Using 200 ul wide-bore pipette tip, carefully transfer DNA thread to a new microfuge tube.
  • Do not centrifuge in this step. Centrifugation precipitates tRNA toegther.
  • To make wide-bore tip, cut the tip by scissor.

    28. Wash DNA pellet by adding 1 ml of 70% ethanol.

    29. Mix by inverting a tube and pipet out DNA thread to a new tube.

    30. Remove ethanol and wash with 70% Et-OH two more times to remove any contaminating tRNAs and salt, etc.


  • Removal of tRNA contamination
    Repeat ethanol precipitation 2-3 times to remove tRNA contamination.

    31.Resuspend DNA pellet in 200~400 ul, H2O or TE buffer depending on DNA pellet size.
  • For small DNA pellet, resuspend in 200 ul H2O.
  • Optional: For large size DNA pellet, keep warm at 37oC for 5 min to resuspend DNA pellet completely.

    32. Add 0.1 vol. 3 M NaOAcpH5.2.

    33. Add 2.5 vol. 100% Et-OH and mix by inverting tube 3~5 times.
  • Do not vortex. tRNA may co-precipitate with plasmid DNA by vortexing.

    34. DNA thread should be formed without centrifugation.
  • Using 200 ul wide-bore pipette tip, carefully transfer DNA thread to a new microfuge tube.
  • Do not centrifuge in this step. Centrifugation precipitates tRNA toegther.

    35. Wash DNA pellet by adding 1 ml of 70% ethanol.

    35. Mix by inverting a tube and move DNA thread to a new tube.

    36. Remove ethanol completely and air dry for 5 min.

    37.Resuspend DNA pellet in 20-400 ul TE buffer.
  • Three times of ethanol precipitation usually removes tRNA completely, resulting sequencing-quality DNA.



    NOTES




    KIT INFORMATION




    REFERENCES


  • Please send your comment on this protocol to "editor@MolecularInfo.com".

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