Difference between revisions of "Team:Tuebingen/Experiments"
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<h4> 3A-Assembly </h4> | <h4> 3A-Assembly </h4> | ||
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+ | <h4> Restriction </h4> | ||
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+ | <h4> Transformation </h4> | ||
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<li>0,2μl EcoRI-HF</li> | <li>0,2μl EcoRI-HF</li> | ||
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<li>Fill up with water to 10μl.</li> | <li>Fill up with water to 10μl.</li> | ||
<li>Run a 1% agarose gel at 37°C for 1,5h.</li> | <li>Run a 1% agarose gel at 37°C for 1,5h.</li> | ||
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</ul> | </ul> | ||
<h4> PCR </h4> | <h4> PCR </h4> |
Revision as of 09:27, 25 August 2015
![](https://static.igem.org/mediawiki/2015/b/ba/Team_Tuebingen_menu_coli_mit_lightstrahl.png)
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Protocols
Prep
Colony-PCR
3A-Assembly
Restriction
Transformation
- 0,2μl EcoRI-HF
- 0,2μl PstI
- 1μl Buffer
- 1μg DNA[?]
- 4μl Plasmid
- 2μl Cre
- Fill up with water to 10μl.
- Run a 1% agarose gel at 37°C for 1,5h.
PCR
Ligation
- 5 μl Water
- 2 μl Puffer NEB
- 1 μl Ligase NEB
- 2 μl Plasmid (pSB)
- 10 μl Insert
- 4 °C o/n
Testrestriktion
Gel extraction
1% Agarose gel
- 3 μl Midori green
- 100 ml TBE
- 1g Agarose
Pouring agar plates
- LB Agar-Agar
- 200ml 1:1000 Chloramphenicol(11 plates)
- 200ml 1:1000 Ampicillin(11 plates)
DNA Purification using Promega Wizard SV Gel and PCR Clean-Up System Kit
This is a modified protocol for the Wizard SV Gel and PCR Clean-Up System Kit from Promega
- Following electrophoresis, excise DNA band from gel and place gel slice in a 1,5ml microcentrifuge tube.
- Add 10μl Membrane Binding Solution per 10mg of gel slice. Vortex and incubate at 50-60°C until gel slice is completely dissolved.
- Insert SV Minicolumn into Collection Tube.
- Transfer dissolved gel mixture to the Minicolumn assembly. Incubate at room temp. for 1 minute.
- Centrifuge at 16,000xg for 1 min. Discard flowthrough and reinsert Minicolumn into Collection tube.
- Add 700μl Membrane Wash Solution (ethanol added). Centrifuge at 16,000xg for 1 min. Discard flowthrough and reinsert Minicolumn into Collection tube.
- Repeat this step with 500μl Membrane Wash Solution. Centrifuge at 16,000xg for 5 min.
- Empty the Collection Tube and recentrifuge the column assembly for 1,5 min.
- Leave the tubes open for 10 min (to let any rest of ethanol evaporate).
- Carefully transfer Minicolumn to a clean 1.5ml microcentrifuge tube.
- Add 30μl of Nuclease-Free-Water (65°C) to the Minicolumn. Incubate at 65°C for 5 min. Centrifuge at 16,000xg for 1 min.
- Discard Minicolumn and store DNA at 4°C or -20°C.
Experiments & Protocols
Describe the experiments, research and protocols you used in your iGEM project.
What should this page contain?
- Protocols
- Experiments
- Documentation of the development of your project