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Team:Colombia Uniandes/Protocols
From 2013.igem.org
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.
The final eluate contains the DNA – Do not discard.
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.
The final eluate contains the DNA – Do not discard.
