Team:Tuebingen/Experiments
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Protocols
MiniPrep
- Resuspension of the bacteria pellet (from 3ml culture) in 600μl H2O.
- Add 100 μl cell Lysis Buffer, invert it.
- Add 350 μl cold Neutralization Solution, invert the mixture.
- Centrifuge 5 min. at max. speed.
- Transfer supernatant in PureYield Minicolumn in Collection Tube.
- Centrifuge for 30 sec, discard the flowthrough.
- Add 200 µl Endotoxin Removal Wash(ERB), centrifuge for 30sec, discard the flowthrough.
- Add 400 µl Column Wash Solution(CWC), centrifuge for 30sec, discard the flowthrough.
- Place column in reaction vessel, add 30 μl Elution Buffer to minicolumn matrix, incubate 1 min at 37°C.
- Centrifuge the mixture.
- Measurement of the DNA concentration at the Nanodrop
Colony-PCR
- Pick a colony from plate and strike out in PCR tubes.
- Add 5μl Q5 High-Fidelity 2X MasterMix from NEB.
- Add 1,8μ of each primer (1μMol): fw/rv
- Add x μl water up to 10μl per mixture.
3A-Assembly
Control Restriction
- 0,2μl EcoRI-HF
- 0,2μl PstI
- 1μl Buffer
- 250ng DNA[?]
- Fill up with water to 10μl.
- Incubate at 37°C for 1,5h.
- Run 1% agarose gel
Transformation
- Take 50μl competent cells and 5μl ligation/plasmid.
- Incubate at 37°.C
- Centrifuge at 2000xg for 2 min.
- Discard the supernatant, resuspend the rest and do the plating.
PCR
Ligation
- 5 μl Water
- 2 μl Puffer NEB
- 1 μl Ligase NEB
- 2 μl Plasmid (pSB)
- 10 μl Insert
- 4 °C o/n
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)
Gel extraction / 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
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