Exeter/3 July 2013

From 2013.igem.org

Making stock solutions of chloramphenicol, ampicillin and kanamycin

Chloramphenicol and kanamycin are TOXIC so need to be carefully weighed out in the fume hood. We are making 10ml stock solutions of all three antibiotics (none of the BioBricks we are using are in a tetramycin plasmid, so we have not made a stock solution of this antibiotic).

- Ampicillin, stock solution is 100 mg/ml, so for 10ml we need 1000 mg (1 g)

- Chloramphenicol, stock solution is 35 mg/ml, so we need 350mg

- Kanamycin, stock solution is 50 mg/ml, so we need 500 mg

Chloramphenicol must be dissolved in ethanol, the others can be dissolved in distilled water.

Weigh each antibiotic into an Eppendorf tube, then transfer into a 10 ml Falcon tube. Add 10 ml ethanol/water using a serological pippette, using a little of the ethanol/water to wash the last of the antibiotic from the Eppendorf. Seal each Falcon tube, and store in the freezer.

Making antibiotic LB agar plates

We will be using the 200 ml Duran bottles of LB agar we made yesterday.

Place the bottle in the microwave (with the lid off!) for approximately 5 minutes. Keep an eye on it, they bubble up very easily. Remove from the microwave every so often and swirl to help the agar dissolve.

For each 200 ml of LB agar, 200 ul of antibiotic stock solution needs to be added. This can only be done when the agar has cooled sufficiently, approximately below 50oC. 200ml of agar should make about 8 plates of normal thickness.

Add the antibiotic to the agar in a fume hood, swirl gently and pour each plate. The plates need to be left in the fume hood for ~20 minutes to solidify.