Difference between revisions of "Team:Tuebingen/Experiments"

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

Inspiration

• 2014 Colombia
• 2014 Imperial
• 2014 Caltech