Making LB Agar Plates

1. Determine how many LB Agar plates you need to make the correct amount of LB Agar. 20mL of LB Agar go on each petri dish. (Ex: 3.5g of LB Broth with Agar + 100mL of DI Water to make about 5 plates)

2. Measure out the determined amount of LB Broth with Agar (Chemical Shelves) using the analytical balance in the main lab.

3. Add the amount of DI water you need and then add the measured out LB Broth with Agar to a bottle.

(Choose the size of the bottle based on how much solution you're making. Ensure there is room in your bottle, so it won't overflow. Ex: If you're making 200mL of LB Agar, you would use a 500mL bottle.)

4. Add a stir bar to the solution and stir/heat the agar on the magnetic stirring hot plate in the main lab. Set the stirring setting as high as it can go where the stir bar is spinning in the middle without clanking the sides. Heat to boiling (about medium heat) and allow the powder to dissolve.

5. Remove the stir bar (using the magnetic rod). Move the bottle to the small autoclave in the shared equipment room (use the insulated gloves!).

6. Autoclave for 30 minutes at 121°C.

7. Retrieve from autoclave and allow the bottle to cool to 65°C. Check with a thermometer usually found in the gel electrophoresis drawer.

8. Grab the desired amount of petri dishes from the main lab (in the back section) and bring them to the Tissue Culture Hood. Spray the hood with ethanol and wipe with a paper towel. Then open the culture plate bag and without touching the inside of the bag gently remove however many petri dishes you need. When done close the bag and return to the drawer in the main lab.

9. Use a sharpie to label the plates with "LB + [antibiotic]" initials and date on the edge of the plate. Ex: "LB +AMP LC 5/26/21"

10. Retrieve the antibiotic corresponding to what bacterial DNA you are transforming. Most antibiotics are in Box 5 in Freezer C. Ex: If you are using pGHE use ampicillin.

11. Allow it to defrost and then add the desired amount to the cooled solution. 100uL of antibiotic per 100mL of solution. Ex: 500uL of spectinomycin for 500mL of LB Agar solution.

12. Swirl the bottle to mix and then pipette ~20mL of solution to each plate. Use the 25mL pipettes in the main lab.

13. Allow the plates to cool without the lids on for about 20 minutes in the tissue culture hood. Check that it is solid to ensure they are finished cooling.

14. Move to the Fridge B to store. Place the agar side up (upside down), so condensation does not affect the LB Agar.