LB agar plates & LB broth

LB agar plates with antibiotic (e.g. Ampicillin) are used for growing / selecting bacteria transformed with plasmids (plasmids containing an antibiotic resistance gene). LB broth is then used to clone / grow plasmid transformed bacteria to high levels for plasmid harvest.

To make LB Broth:

1. Add the following to 800mL of sterile deionised water:
10g Bacto-tryptone for a final concentration of 1%
5g yeast extract for a final concentration of 0.5%
10g NaCl for a final concentration of 1%

2. Adjust volume to 1L with sterile deionised water and mix

3. Adjust pH to 7.0 - 7.5 with 5M NaOH

4. Sterilize by immediately autoclaving at 121°C for 25 minutes

5. Store at room temperature (if no antibiotic is added).

6. Store at 4°C in the dark if antibiotic is added.
For Ampicillin (Amp) add for a final concentration of 100ug/mL.(Add antibiotic below 55oC after autoclaving. Ideally antibiotic should be added just prior to use).

LB Agar plates

1. Prepare LB broth as above adding 15g/L agar (1.5%) and sterilize by autoclaving at 121°C for 25 minutes. (If making LB agar plates from LB broth stock: For 10 plates use 250mL of broth with 3.75g agar and sterilize by autoclaving at 121°C for 25 minutes).

2. Cool to below 55°C, add antibiotic and pour into plates in laminar flow cabinet. For Ampicillin (Amp) add for a final concentration of 100ug/mL. (For 10 plates use 250uL of 1000x Amp stock with 250mL of LB agar mix. 1000 X Amp stock can be made by adding 100mg to 1mL sterile deionised water. Store 1000 X Amp stock at 4oC or -20oC in the dark).

3. Let plates set then invert to prevent liquid build up on the agar, write the date on each plate, wrap in clingwrap and store at 4°C in the dark for approximately 1 month if not using immediately.