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Morning: agar plates
We have prepared 5 agar plates with 50 µg/ml of Spectinomycin and 5 with 100 µg/ml. One of each has been kept open, to dry for today's transformation.
We have used two bottles with 150 ml of medium each. That means, per bottle:
- 3 g of LB
- 1.5 g of agar
- 150 ml of di water
Our spectinomycin has a concentration of 100 mg/ml. After autoclaving, we have added 75 µl of spectinomycin to one bottle (50 µg/ml) and 150 µl to the other (100 µg/ml).
Protocol: Agar LB plate preparation
Ingredients:
- LB: 20 g/L
- Agar: 10 g/L (Note: not agarose)
- di water
Procedure:
- Autoclave all the ingredients together in a glass bottle, loose cap and covered with alu foil, with 20 min liquid program (106). This takes about 1h in the autoclave.
- Prepare the plates by labeling and ethanol cleaning a big enough surface.
- Close the bottle and allow to cool down to 50-55ºC (colder will start to solidify, warmer might degrade the antibiotic).
- Add the antibiotic to the bottle: 100 µg/ml for ampicillin, 50-100 µg/ml for spectinomycin.
- Add 25-30 ml of the medium with antibiotic to each plate. Leave half open for about 2 h for cooling.
- Close hte plates. Wrap the in alu foil (antibiotics are sensitive to light) and store the at 4ºC.
- Comments
For some reason one of the bottles showed a slightly darker color (the 100 µg/ml one).
Afternoon: transformation
Using the DNA, sample #1, prepared the 29 June. Chosen because it looked the nicest in the gel the 5th July. 2 tubes prepared, adding 2 µl of DNA and 50 µl of cell culture.
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)
