Team:UNICAMP-EMSE Brazil/protocols/Culture

Culture Media:

• 500 mL of milli Q water
• 10 g tryptone
• 5 g yeast extract
• 5 g NaCl
• Solubilize the solids, correct the pH to 8,0. Complete the volume to 1 L with milli Q water.
• Autoclavate.

Solid LB:

• Add 15 g/L of agar in the liquid LB media.
• Autoclavate.

Pouring Plates:

• Turn on the flux
• Wait until the solid media cools down after autoclaving (you should be able to hold it, but needs to be still warn). If the media has already solidified, you can melt it in the microwave oven (don’t let it boil) and wait it to cool down.
• Add the specific antibiotic in the media corresponding to the final concentration:

• Ampicillin: 50 μg/mL
• Tetraciclin: 12 μg/mL
• Kanamicin: 30 μg/mL
• Clorafenicol: 20 μg/mL

• Using sterile technique, pour the media into the plates. Cover the base of the plate, and then just a bit more after that.
• Recap each plate upon pouring. If there are lots of bubbles in your plates (i.e., more than one or two on the edge), you can flame the plate using the small bunsen burner to eliminate bubbles. Another way to remove the fine bubbles that may be in your flask before pouring is to mist the inside of the flask with a 75% ethanol spray bottle.
• Leave plates to dry and cool for a while (overnight even).
• Label the bags following taping rules:

• Name of the media, antibiotic and concentration used, bacteria, plasmid, gene cloned, date and name of the person who done it.

• After dried, store in the refrigerator.