Chromosomal DNA isolation from E. coli protocol

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Solutions/reagents:

Equipment:

  • Centrifuge
  • Incubator

Steps:

  1. Measure out 1 volume ice-cold Killing Buffer into culture grown in your favorite medium.
    Vortex the mixture for a few secs.
    Store the tube on ice.
    Samples should be processed as fast as possible.
  2. Centrifuge at maximum speed for 3 mins at 4°C, gently aspirate out the supernatant and discard it.
  3. Measure out 300 µl of TE into Eppendorf tube (1).
    Add 40 µl of 10% SDS.
    Add 3 µl of 0.5M EDTA.
    Resuspend pellet by vortexing/by shaking vigorously.
  4. Incubate at 65°C for 5 mins.
  5. Add 750 µl of isopropanol.
    Vortex the mixture for a few secs.
  6. Centrifuge at maximum speed for 5 mins at room temperature, gently aspirate out the supernatant and discard it.
  7. Add 500 µl of TE.
    Add 2 µl of RNase A.
    Resuspend pellet by vortexing/by shaking vigorously.
  8. Incubate at 65°C for 30 mins.
  9. Add 2 µl of proteinase K.
    Incubate at 37°C for 15 mins.
  10. phenol extract (2x phenol & 2x chlorophorm).
  11. Add 1 ml of ethanol.
    Add 40 µl of 3M Na-Acetate.
    Store at room temperature for 12 hrs(overnight).
  12. Centrifuge at maximum speed for 15 mins at 4°C, gently aspirate out the supernatant and discard it.
    Add 50 µl of distilled water.
    Resuspend pellet by vortexing/by shaking vigorously.

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 13 hrs, 13 mins

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