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E.Coli transformation with pGL4.30, pMP and melanopsin plasmid
Aim: Amplify all the plasmids we need for cloning LovTAP into pMP and the Fussenegger parts.
3 plasmids:
- pHY42: melanopsin sent by Fussennegger in a piece of paper. It was put in water and 20 µl of DNA were recovered.
- pMP: Mammalian expression plasmid frequently used in LBTC lab.
- pGL 4.30: diluted (1 µl in 19 µl of water). For readouts for the Fussenegger experiment.
We added 50 µl of bacteria to 2 µl of DNA in the 3 cases.
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)
- Comments
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