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Team:Colombia Uniandes/Protocols
From 2013.igem.org
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<li>500 µL of Wash Solution Concentrate (+ ethanol) --> 15000 rpm x 3 min – Discard the collection tube and place the column in a new one. | <li>500 µL of Wash Solution Concentrate (+ ethanol) --> 15000 rpm x 3 min – Discard the collection tube and place the column in a new one. | ||
<li>30 µL of water (miliQ) --> 14000 rpm x 1 min.</li> | <li>30 µL of water (miliQ) --> 14000 rpm x 1 min.</li> | ||
| - | <li>30 µL of water (miliQ) | + | <li>30 µL of water (miliQ) --> 14000 rpm x 1 min.</li> |
| - | <li>The final eluate contains the DNA – | + | <li>The final eluate contains the DNA – Do not discard!</li> |
Revision as of 02:51, 23 September 2013
Protocol for extraction of yeast genome:
GenElute DNA Kit from Sigma-Aldrich with modifications
- 1,5 mL 2 min x 15000 rpm.
- 100 µL zymolyase solution.
- Vortex.
- Incubate 37°C x 30 min.
- 180 µL of Lysis Solution T.
- 20 µL of Proteinase K.
- Incubate 37°C x 30 min.
- 200 µL of Lysis Solution C.
- Vortex x 15 s.
- Incubate 55°C x 10 min.
- Column Preperation --> 500 µL Column Preparation Solution --> 13000 rpm x 1 min – Discard the eluate.
- 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.
- 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.
- 500 µL of Wash Solution Concentrate (+ ethanol) --> 15000 rpm x 3 min – Discard the collection tube and place the column in a new one.
- 30 µL of water (miliQ) --> 14000 rpm x 1 min.
- 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
- 1,5 mL 2 min x 15000 rpm.
- 100 µL zymolyase solution.
- Vortex.
- Incubate 37°C x 30 min.
- 180 µL of Lysis Solution T.
- 20 µL of Proteinase K.
- Incubate 37°C x 30 min.
- 200 µL of Lysis Solution C.
- Vortex x 15 s.
- Incubate 55°C x 10 min.
- Column Preperation --> 500 µL Column Preparation Solution --> 13000 rpm x 1 min – Discard the eluate.
- 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.
- 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.
- 500 µL of Wash Solution Concentrate (+ ethanol) --> 15000 rpm x 3 min – Discard the collection tube and place the column in a new one.
- 30 µL of water (miliQ) --> 14000 rpm x 1 min.
- 30 µL of water (miliQ) --> 14000 rpm x 1 min.
- The final eluate contains the DNA – Do not discard!
