Team:Tuebingen/Experiments

Experiments

Results

Design

Parts

Safety

Interlabstudy

Notebook

Collaboration

References

SynBio-Day

SchoolClass@lab

Theatre Freiburg

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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, vortex the mixture.
Centrifuge 5 min. at max. speed.
Transfer supernatant in PuteYield Minicolumn in Collection Tube.
Centrifuge for 15 sec, discard the throughflow.
Add 200 µl Endotoxin Removal Wash(ERB), centrifuge for 15sec, discard the throughflow.
Add 400 µl Column Wash Solution(CWC), centrifuge for 15sec, discard the throughflow.
Place column in reaction vessel, add 30 μl Elution Buffer to minicolumn matrix, incubate 1 min at 37°C.
Centrifuge the mixture.
Messurement of the DNA concentration at the Nanodrop

Colony-PCR

Centrifuge 200μl o/n culture, discard the supernatant and put the pellets in PCR tubes.
Add 5μl Q5 High-Fidelity 2X MasterMix from NEB.
Add 1,8μ of each primer: fw/rv
Add x μl water up to 10μl per mixture.

3A-Assembly

Restriction

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.

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.