- 250 mL (makes about 10 plates)
- All temperatures are centigrade.
- Jon’s trick: make up 250 mL in a 600 mL BEAKER (not flask). This makes pouring easy. No volume is lost from the beaker during autoclaving.
- 3.75 g agar (NOT agarose!)
- 6.25 g LB
- Water up to about 250 mL (you can use the inaccurate gradations on the beaker).
- Add agar and LB. They will mix during autoclaving. Cover beaker with foil. You may add a stir bar at this point to stir in the ABs later (I, Jon, simply swirl them to mix). Use a stir bar designated for the autoclave, since heat ruins magnets over time.
- Autoclave 30 minutes.
- Label the plates on the bottom w/ AB and date. Use the 100×15 mm plates – the 100×20 plates are for dicty. If you are not using a water bath you must have this done in advance lest the agar set prematurely.
- Use a water bath at 45 for about 30 minutes, or wait til agar gets to about 50 degrees on the bench. 50 degrees is very hot but not painful to touch. You can press a thermister to the outside of the beaker to get the approximate temperature.
- Add the antibiotics when the agar is less than 50 degrees. Hi temps can break down ABs. We store the ABs in a freezer box at -20 in 1000-fold stocks. So add 250 uL of AB stock to 250 ML agar. Thaw the ABs in advance, no later than when the agar is cooling so they are thawed when you need them. Otherwise the agar might set before you are ready. Mix or swirl-in the ABs.
- Pour agar into plates up to about where the line is.
- Bust bubbles if necessary by flaming with a Bunsen burner.
- Spread plates out to cool them.
- Wrap them in plastic bags, also labeled with date and AB.
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