- 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.