Team:Colombia Uniandes/Protocols
From 2013.igem.org
(Difference between revisions)
(→GenElute DNA Kit from Sigma-Aldrich with modifications) |
(→Protocol for extraction of yeast genome:) |
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<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>The final eluate contains the DNA – Do not discard!</li> | <li>The final eluate contains the DNA – Do not discard!</li> | ||
+ | </ol> | ||
+ | ===Protocol for electrocompetent cells=== | ||
+ | |||
+ | <ol> | ||
+ | <li>Divide the ON culture in 50 mL falcon tubes.</li> | ||
+ | <li>Centrifuge 8000 rpm x 10 min.</li> | ||
+ | <li>Discard supernatant.</li> | ||
+ | <li>Resuspend everything with water in two falcons.</li> | ||
+ | <li>Centrifuge again.</li> | ||
+ | <li>Discard supernatant and resuspend with water. Wash with water two more times.</li> | ||
+ | <li>Centrifuge again.</li> | ||
+ | <li>Discard supernatant.</li> | ||
+ | <li>Resuspend with glycerol with water. Glycerol 10%.</li> | ||
+ | <li>Centrifugue.</li> | ||
+ | <li>Repeat steps 8-10.</li> | ||
+ | <li>Discard supernatant and divide what is left in eppendorfs.</li> | ||
+ | </ol> | ||
+ | CAUTION: Everything must be done on ice (0-3°C) (reactants, centrifuge, transport, containers). |
Revision as of 02:55, 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 electrocompetent cells
- Divide the ON culture in 50 mL falcon tubes.
- Centrifuge 8000 rpm x 10 min.
- Discard supernatant.
- Resuspend everything with water in two falcons.
- Centrifuge again.
- Discard supernatant and resuspend with water. Wash with water two more times.
- Centrifuge again.
- Discard supernatant.
- Resuspend with glycerol with water. Glycerol 10%.
- Centrifugue.
- Repeat steps 8-10.
- Discard supernatant and divide what is left in eppendorfs.
CAUTION: Everything must be done on ice (0-3°C) (reactants, centrifuge, transport, containers).
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 electrocompetent cells
- Divide the ON culture in 50 mL falcon tubes.
- Centrifuge 8000 rpm x 10 min.
- Discard supernatant.
- Resuspend everything with water in two falcons.
- Centrifuge again.
- Discard supernatant and resuspend with water. Wash with water two more times.
- Centrifuge again.
- Discard supernatant.
- Resuspend with glycerol with water. Glycerol 10%.
- Centrifugue.
- Repeat steps 8-10.
- Discard supernatant and divide what is left in eppendorfs.