Molecular Techniques and Methods

Isolation of Total RNA from Bacterial Cells

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION




MATERIALS AND SOLUTIONS

0.12 M Sodium Phosphate Buffer (pH 7.2) (100 ml)
0.12 M Sodium Phosphate Buffer ---------- 12 ml of 1 M Sodium Phosphate Buffer
Deionized H2O ---------------------------- 88 ml


Proteinase K (1 ml)
0.5 mg Proteinase K ----------------------- 25 ul of Proteinase K (20 mg/ml)
Deionized H2O ---------------------------- 975 ul
  • Store at -20oC.


    6 M Guanidine thiocyanate (100 ml)
    Guanidine thiocyanate ---------------------------- 70.9 g
    Add deionized H2O to make a final volume of ---- 100 ml
  • Store at 4oC.


    Acid phenol: chloroform: IAA (25: 24: 1)




    PROCEDURES

  • All manipulations should be performed on ice! Always wear gloves!



  • 1. Inoculate bacteria into 10 ml of LB and grow overnight at 37oC.

    2. Use 5 ml of this culture to inoculate 50 ml of LB and grow for 2.5 hours at 37oC, to an absorbance A600 of 0.5-0.6.

    3. Centrifuge a 1.5 ml culture in a microfuge tube at 13,000g for 2 minutes.

    4. Resuspend the pellet in 300 ul of 0.85% NaCl.

    5. Centrifuge at 13,000g for 2 minutes and discard the supernatant.

    6. Resuspend the pellet in 400 ul of 0.12 M Sodium Phosphate Buffer (pH7.2).

    7. Add 50 ul of 5% SDS and 50 ul of Proteinase K (0.5 mg/ ml).

    8. Vortex and incubate at 37oC for 20 minutes.

    9. Add 750 ul of 6 M Guanidine thiocyanate.

    10. Vortex and centrifuge at 13,000g for 3 minutes.

    11. Transfer the supernatant to a fresh tube.

    12. Extract sample with Acid phenol: chloroform: IAA.

    13. Add 0.1 volume of 3 M Sodium acetate (pH 7.0) and 2.5 volume of cold ethanol.

    14. Precipitate RNA at -20oC for 1 hour.

    15. Centrifuge at 13,000g for 20 minutes and discard the supernatant.

    16. Wash the pellet with 70% ethanol by resuspending and centrifuging at 13,000g for 10 minutes.

    17. Air dry the pellet.

    18. Dissolve the pellet in 10-100 ul TE (pH 7.0).




    NOTES

  • The isolation procedure can be scaled up by increasing the components proportionately.


  • Do not vacumm dry the RNA pellet. It will be very difficult to resuspend the RNA pellet.





  • KIT INFORMATION




    REFERENCES

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

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