Team:Queens Canada/agar
LB Broth and Agar Plates
Project: QGEM Team
Authors: Ellis Kelly
Date: 2018-05-22
Tuesday, 5/22/18
LB Broth (1 L) - 2 bottles of 500mL each, with Don's Media
Materials:
10 g Bacto-Tryptone
5 g Bacto-Yeast Extract
10 g NaCl
1 L ddH2O
Procedure:
Add 900mL of ddH2O to a large beaker with a stir bar. Add Tryptone, Yeast Extract, NaCl. Measure the pH with indicator strips. If the pH is 7.0, add another 100 mL of ddH2O.
** When using Tryptone, wear a face mask and goggles. It is dry and very powdery and dangerous to inhale.
Add the mixture to 2X 500mL glass bottles. Add autoclave tape and autoclave for 30 min. ******MAKE SURE TO LOOSEN THE LIDS BEFORE AUTOCLAVING
LB Agar Broth (1L):
Materials:
10 g Bacto-Tryptone
5 g Bacto-Yeast Extract
10 g NaCl
ddH2O
15 g of Bacto-agar
Procedure:
Add 900mL of ddH2O to a large beaker with a stir bar. Add Tryptone, Yeast Extract, NaCl and agar. Measure the pH with indicator strips. If the pH is 7.0, add ddH2O until the total volume is 1L.
Add to a 1L glass bottle, pour in a little at a time and then swirl to ensure agar stays in solution. Autoclave.
Preparing Ampicillin:
Materials:
1 g of Ampicillin
ddH20
15mL tube
autoclaved tubes
Procedure:
Weigh out ampicillin on weigh sheet. Add to 15mL tube. Add ddH2O until volume reaches 10 mL. Add a few mL's at a time and then swirl to ensure the ampicillin dissolves.
Aliquot final liquid into 500uL tubes. Store at -20C.
Preparing Ampicillin Plates:
Materials:
LB Agar Broth
Ampicillin
Plastic plates
Procedure:
Let the LB agar broth cool until it is warm, but safe to touch (too hot and the ampicillin will be ineffective, too cool and you will have difficulty pouring the plates).
Obtain 1000uL of ampicillin and let thaw.
Start a flame. Keep all the materials within close proximity to the flame.
Add the ampicillin to the LB agar broth. Shake.
Slowly pour the agar into the plates until the bottom is just covered. Flame any bubbles - not too long otherwise the ampicillin will be ineffective.
Let the plates cool for a few minutes with the lid half covering the plate. This prevents too much condensation from occuring.
After a few minutes, put the lid on fully.
Let the plates set overnight and store at -4C.