Team:Colombia Uniandes/Protocols

Protocol for extraction of yeast genome:

GenElute DNA Kit from Sigma-Aldrich with modifications

1. 1,5 mL 2 min x 15000 rpm.
2. 100 µL zymolyase solution.
3. Vortex.
4. Incubate 37°C x 30 min.
5. 180 µL of Lysis Solution T.
6. 20 µL of Proteinase K.
7. Incubate 37°C x 30 min.
8. 200 µL of Lysis Solution C.
9. Vortex x 15 s.
10. Incubate 55°C x 10 min.
11. Column Preperation --> 500 µL Column Preparation Solution --> 13000 rpm x 1 min – Discard the eluate.
12. 200 µL of ethanol (100%) to the lysate – Vortex. --> Transfer the entire contents of the tube into the binding column --> 12000 rpm x 1 min --> Discard the collection tube and place the column in a new one.
13. 500 µL of Wash Solution 1 to the column. --> 12000 rpm x 1 min --> Discard the collection tube and place the column in a new one.
14. 500 µL of Wash Solution Concentrate (+ ethanol) --> 15000 rpm x 3 min – Discard the collection tube and place the column in a new one.
15. 30 µL of water (miliQ) --> 14000 rpm x 1 min.
16. 30 µL of water (miliQ) --> 14000 rpm x 1 min.
17. The final eluate contains the DNA – Do not discard!

Protocol for electrocompetent cells

1. Divide the ON culture in 50 mL falcon tubes.
2. Centrifuge 8000 rpm x 10 min.
3. Discard supernatant.
4. Resuspend everything with water in two falcons.
5. Centrifuge again.
6. Discard supernatant and resuspend with water. Wash with water two more times.
7. Centrifuge again.
8. Discard supernatant.
9. Resuspend with glycerol with water. Glycerol 10%.
10. Centrifugue.
11. Repeat steps 8-10.
12. Discard supernatant and divide what is left in eppendorfs.

CAUTION: Everything must be done on ice (0-3°C) (reactants, centrifuge, transport, containers).