From Protists

LB-Kanamycin Plates

1. Turn on water bath to 42C setting.
2. Take the Kanamycin (10mg/mL) from the -20C freezer (5mL in 15mL tubes in the basket) and thaw it in the water bath (with cap wrapped in parafilm) or thaw at room temp. Vortex it occassionally to thaw it faster. When done thawing, place it in 4C until needed.
3. Make sure to wear gloves from this step on. While Kanamycin is thawing, add 40g of LB Agar (in first chemical cabinet) to 1L of distilled water in a 2L-flask. Cover the opening with aluminum foil. Swirl to mix. (don't worry if all of it does not go into solution. This will happen in the autoclave.)
4. Autoclave on liquid cycle for 45 minutes. Fill the autoclave tub up with ~1 inch of water before autoclaving.
5. After removing it from the autoclave, cool it in the 42C water bath for ~10 minutes. During this cooling period, set up empty plates under the 2 hoods after wiping down w/ ethanol.
6. When the bottom of the flask is not extremely hot to the touch through your gloves after swirling, add 5mL of Kanamycin (10mg/mL) to the flask under the hood. Swirl to mix. Let the bubbles settle somewhat before pouring.
7. Pour plates under the hoods. Make sure that the agar is not too bubbly and that you cover the entire bottom of the plate. Place the top of each plate partially over the cooling bottom to cover part of the plate (controls contamination). Do not cover the plate completely with the top until it has fully cooled (condensation on the top of the plate can drip onto the agar and contaminate the plate). The first plates should be cool after you finish pouring the last plates and start the cleaning process for the 2-mL flask.
8. Put the plates back in the sleeve. Make sure the plates are placed in the sleeve agar-side up. Tape the sleeve closed and label the tape LB-Kan, the date, and your initials. Store in the 4C walk-in with the plates agar-side up.