Making LB Plates

October 26, 2021 - Resuspended 10ng plasmid in 100µL MBG water to make a concentration of 100ng/uL

November 7, 2021 - Making LB Agar Plates

1. Measured out 7g of LB Broth with Agar (using analytical balance in main lab) and 200mL of DI Water

2. Poured both into a 500mL bottle

3. Added a stir bar and heated solution

   1. Stir setting = highest setting without hitting the sides

   2. Heat setting = to boiling (~medium) , we did not bring the solution to a full boil.

   3. Allowed powder to dissolve

4. When dissolved, removed stir bar using magnetic rod.

5. Autoclaved in the small autoclave for 30 minutes at 121ºC

   1. As autoclave starts to cool, started setting up hood.

6. Allowed bottle to cool to 63ºC

   1. Checked with thermometer

7. Got 10 petri dishes and brought them to the tissue culture hood

8. Sprayed hood with ethanol and wiped down to sterilize

   1. We also sprayed the bag of plates, the thermometer, the bottle of broth, and the pipette with ethanol before bringing it into the tissue culture hood.

9. Without touching the inside of the bag, gently removed the 10 dishes

   1. Closed bag and return to drawer when finished

10. Labeled the plates

    1. “LB + AMP MN AJH 11/7/21”

11. Retrieved ampicillin from Box 5 in Freezer C

12. Allowed ampicillin to defrost then added 200µL of ampicillin to the solution

    1. Swirled bottle to mix

13. Pipetted ~20mL of the agar solution into each plate

    1. Used 25mL pipettes in main lab

14. Allowed plates to cool without the lids for at least 20 minutes in tissue culture hood

    1. Checked that they are solid

15. Moved to Fridge B for storage agar side up