This recipe is designed to make 1 litre of Luria-Bertani (LB) media for growing E. Coli with standard plasmids. To watch the video of this protocol click here.
10 g Bacto-Tryptone
5 g Bacto-Yeast extract
10 g NaCl
Up to 1 litre with water
Potential Hazard: Powders can be sensitising and you can develop allergies to them. Avoid inhalation of aerosolised powder.
Measure out the amounts above and add to a 1 litre bottle with a good working lid (see note below). Fill with clean distilled water (ideally at least 18 megaohms). Tighten lid and shake to mix the liquid and powder, don’t expect to dissolve it all but simply to free the powder from the bottom and remove the major clumps. If not mixed properly the powder can bake on the bottom of the bottle. Undo the lid about half a turn; add some autoclave tape and then autoclave.
After autoclaving make sure the lid is tightened back up. Ideally the lid is tightened whilst the bottle is warm/hot as this will create a vacuum as the liquid cools. When cool add the correct amount of antibiotic (see below) and store at 4ºC. It can be left in the cold room for a few months but watch out for fungus contamination and a decline in antibiotic activity over time. To maintain sterility it is best to add the antibiotic and take aliquots out of the bottle in a class 1 or 2 laminar flow hood.
Notes and Tips:
Tip 1: Many lab bottles have a blue or clear plastic rim which can go missing or get burnt by flaming. These blue rims prevent the liquids running down the bottle when pouring and keep an air tight seal after autoclaving. Make sure the rim is intact.
Tip 2: 1 litre is quite a lot of media and antibiotic, scale down the recipe to suit your needs. I generally make 0.5 litres at a time.
Note 1: Antibiotic concentrations
Dissolved and Stored in Water: add 1µl per 1ml of media
Kanamycin – Stock 50mg/ml, final concentration 50µg/ml
Ampicillin - Stock 100mg/ml, final concentration 100µg/ml (50µg/ml can also be used, but more satellite colonies will appear)
Streptomycin – Stock 50mg/ml, final concentration 50µg/ml
Spectinomycin - Stock 50mg/ml, final concentration 50µg/ml
Dissolved and Stored in Ethanol: Add 1µl per ml media
Chloramphenicol - Stock 25mg/ml, final concentration 25µg/ml
Dissolved and Stored in Methanol: Add 1µl per ml media
Tetracycline HCL - Stock 10mg/ml, final concentration 10µg/ml
Note 2: Pre-mixed LB powders are available from many suppliers and don’t actually work out much more, if any, expensive.
Note 3: For larger plasmids (>50Kb), growing competent cells or cosmids, a richer broth such as 2xYT may be more useful.
Author: Dr Ryan Cawood