Media and Plates¶
How the lab's liquid media, antibiotic stocks, agar plates and competent-cell solutions get made, in the order you would make them. Each step links to its protocol in the Protocol Builder, which does the arithmetic for your batch size, and has a video of it being done in B144.
Pouring plates uses a Bunsen burner and is supervisor-only; see Plate Pouring. Re-melting agar needs the Microwave training.
1. LB agar¶
Bottles of LB agar, sterilized in the pressure cooker. They set solid and keep until you need plates.
2. 2YT broth¶
The liquid medium for growing cultures and for the rescue step after a transformation. Made the same way as LB agar, without the agar.
3. Antibiotic 1000× stocks¶
One video per antibiotic. Weigh roughly 50 mg, record the mass you actually got, and let the protocol work out the solvent volume from it. Choose the antibiotic in the protocol and it shows the matching video.
4. Re-melting agar¶
A set bottle of LB agar goes back into the microwave in short bursts. It boils over fast.
5. Pouring plates (supervisor-only)¶
Cool the agar, add the antibiotic, pour, and mark the plates by antibiotic.
6. Sealing plates with parafilm¶
Sealing slows drying; it does not stop it. Seal fresh plates (or close them in a bag), store them upside-down at 4 °C, and label the bag with the antibiotic and the pour date. Carbenicillin plates are good for 4 weeks from that date. The same wrap goes on any plate you keep after picking colonies. See Plate Hygiene and Freshness.
7. TSS¶
The solution competent cells are made in and frozen in — LB with PEG, DMSO and magnesium. Made once a year in a large batch; a litre serves roughly 20 L of culture.
Never autoclave it. It precipitates, and the batch looks fine until the cells do not transform a week later. Filter sterilise only, and set the pH to 6.5 before you do, because afterwards you cannot put a probe in it. If your filter is not DMSO-resistant — cellulose nitrate is not — filter first and add the DMSO to the sterile filtrate.
8. 5× KCM¶
The salt mix added to a thawed aliquot of competent cells just before the DNA. Unlike TSS, this one autoclaves fine — there is no PEG or DMSO in it.
Weigh from the right bottle: the protocol gives hydrated and anhydrous masses, and using the hydrated figure out of an anhydrous bottle gives the wrong molarity.
New plates need controls¶
Plates poured with anything other than carbenicillin are unproven until a control grows on them. See Control Strains and Plasmids.