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Transformations
Protocol: E.Coli Transformation
- Thaw the competent E.coli (DH5alpha) cells on ice (not in hands!)
- As soon as it is thawed, add 50µl of the cells to the DNA (~50-100 ng of pure plasmid, or some 2 µl usually)
- Let it rest on ice for 20-30 min. Meanwhile, put agar plate (with correct antibiotic) at 37°C for prewarming.
- Put the tube with DNA+E.coli at 42°C for 45 sec - 1 min (heat shock)
- Add 400 µl of LB broth and place at 37°C for 20-30 min (shaking)
- Spread the cells on the prewarmed plate (and let it dry)
- Incubate the plate upside-down at 37°C for ~14-15 hours (leaving it more than 16h decreases the plasmid quality)
The aim was to test our competent cells (by comparing their transformation efficiency with the ones from the lab), and also to find out which spectinomycin concentrations were adapted to cell growth. Thus, three setups were made:
1. Our competent cells transformed with Amp-LovTAP, plated.
2. Competent cells from the lab with Spc-RO (Spectinomycin), plated
3. Lab competent cells with Spc-RO for experiments with the antibiotic concentration. These underwent an overnight culture in 3ml tubes. We made 6 different Spc concentrations: 0, 10, 50, 75, 100, 200 µg/ml.
