IGEM:UNAM/2009/Notebook/IGEM project/2011/05/31

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Extract the plasmid PBBMRCS-5

Abstract

  • Today I extracted plasmid PBBMRCS-5 with the method of alcaline lysis.

Later I did a agarose gel of the plasmid PBBMRCS-5, with the purpose of seeing if the extraction was cleaned of chromosome.

Method to extract plasmid DNA

  • 1. Prepare two eppendorf tubes of Escherichia Coli DH5-α.
  • 2. Centrifuge the tubes to 13 rpm during three minutes and decant the supernatant.
  • 3. Add TE 10, shake the tubes with the vortex and centrifuge.
  • 4. Remove the supernatant with a syringe.
  • 5. Put 100 μl of the solution 1 to he cells and shake it.
  • 6. Put 200 μl of the solution 2 and mix for inversion and centrifuge for ten minutes to 13 mil rpm.
  • 8. Put 200 μl of the solution 3 and shake for inversion.
  • 9. Pass the supernatant to a new eppendorf tube.
  • 10.Put 1 ml of ethanol to 100% in the new eppendorf tube and shake with the vortex.
  • 11.Centrifuge to 13 mil rpm for ten minutes and remove the liquid with the syringe.
  • 12.Put 1 ml of ethanol to 70% and shake with the vortex.
  • 13.Detach the cell pill of the tube and centrifuge.
  • 14.Dry the the cell pill with the speed vac.
  • 15. Put 20 μl of TE-RNASA.

Gel of the plasmid PBBMRCS-5

  • Gel of 1 gr. of agarose and 100 ml of TAE.
Line 1st -> Green ladder 5 μl.
Line 2nd -> PBBRMCS-5 5 μl + 2 μl of dye. (1)
Line 3rd -> PBBRMCS-5 5 μl + 2 μl of dye. (2)
Line 4th -> PBBRMCS-5 5 μl + 2 μl of dye. (3)
Line 5th -> PBBRMCS-5 5 μl + 2 μl of dye. (4)
Line 6th -> Green ladder 5 μl.