Epicentre 1-1
Genomic DNA Extraction with Epicentre Kit
(This can be done on a clean bench top or in the soil hood. Always wear gloves.)
1. Grow an overnight culture of cells to about a concentration of about 0.5x106 3x106 cells
2. Clean down all the work area surfaces with 10% bleach, DNA Away, and 70%
Ethanol.
3. Split your culture into even 5 to 10mL aliquots in 15mL sterile falcon tubes.
4. Centrifuge the culture tubes at 4oC and 4000 rpms for 15 minutes.
5. While cultures are spinning mix together the Lysis Solution.
a. For each spun down culture tube:
b. Add 1uL of the 50ug/uL Proteinase K into 300uL of Tissue and Cell Lysis
Solution.
6. After the cells are spun down, carefully pouring off the supernatant and discard.
a. This should leave approximately 25-50uL of liquid.
7. Vortex the cell pellet to resuspend it into the small volume left.
a. It is ok if the pellet does not completely resuspend at this point.
8. Add 301uL of the Lysis Solution made in step 3 to each tube.
9. If needed pipette the mixture to ensure that the cells are completely resuspended,
10. Transfer the cell mixture into a sterile 1.5mL tube.
11. Incubate the cell mixture at 65oC for 15 minutes
12. Vortexing every 5 minutes.
13. Move the cell mixture to a 37oC water bath for 1-2 minutes, so that the cell
mixture is cooled to 37oC.
14. Add 1uL of the 5ug/uL RNase A to each tube and gently mix everything together.
15. Incubate the tubes at 37oC for 30 minutes.
16. Move the tubes to ice for 3-5 minutes.
17. Add 150uL of MPC Protein Precipitation Reagent to each tube
18. Vortex for 10 seconds to mix.
19. Centrifuge the tubes at 13krpms for 10 minutes to remove the debris.
20. Carefully transfer only the supernatant to a sterile 1.5mL tube.
21. Add 500uL of isopropanol to the supernatant
22. Invert 30-40 times.
23. Pellet the DNA by centrifuging the tubes for10 minute at 4oC and 13krpms.
24. Carefully pour off the isopropanol without dislodging the pellet.
25. Without dislodging the DNA pellet, carefully wash the pellet with 500uL of 75%
ethanol
a. If the pellet becomes dislodged centrifuge the tube for 2 minutes at 4oC
and 13krpms.
26. Pipette off the solution and discard.
27. Repeat step 19 and remove all the residual ethanol by pipetting.
28. Leave the tube open for 5 minutes at room temperature to air dry.
29. Add 35uL of sterile dH2O to resuspend the DNA and save sample at –20oC.