NOTE: You do not need to wear gloves while pouring the agar plates, since no mutagenic chemicals or bacteria are involved in this procedure. I have given you directions to make 300 mL but this can be scaled to any volume.
1. Make 300 mL of the appropriate medium without antibiotic. Bring the volume to 300 mL, pour this into a 1 liter flask, add BactoAgar (1.5% w/v), and cover it with foil and a small piece of autoclave tape. Then autoclave the medium to sterilize it. (See me for directions on autoclaving.)
2. When the autoclaving is done, run cold tap water over the flask while swirling it to cool the molten agar medium. When it is just beginning to be cool enough to put your hand on it for a long time (but there should not be any chunks), it is cool enough to add the antibiotic, IPTG, and X-Gal, if necessary. These last three solutions must be kept sterile so do not contaminate them.
3. Think about these important points in pouring
a petri plate before doing it:
a) You must work quickly, because once the container of agar is below 45 C, it will start to harden within 2-3 minutes.
b) When pouring into the petri dish, pour just enough to cover the bottom; this should fill the dish about half full (or half empty for the pessimists).
c) Although you must work fairly quickly, pour the agar gently to minimize the number of bubbles (bubbles look amazingly similar to bacterial colonies when the agar hardens). There is a trick to help with this problem and I will show you should the need arise.
4. When you are ready to pour and have labeled
dishes in stacks of 5:
a) Open the cover of the petri dish halfway and keep the lid pointed down.
b) Pour the agar to just cover the bottom of the dish. Try to minimize the introduction of bubbles.
c) Repeat for all the dishes and keep them in stacks of five. Do not let the agar slosh up on the sides or lid.
d) Immediately rinse the flask with warm water to facilitate washing the flask.
e) Lable the top dish in each stack and allow to cool.
5. Let the plate harden 15 minutes before moving them. These plates will be stored "upside down".
For each plate add:
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