(Luria-Bertani Broth with 50ug/ml AMPICILLIN).

1 Liter of Luria-Bertani (LB) Broth + Ampicillin

Weigh out:

• Tryptone (Difco 0123) 10.0g
• Yeast Extract (Difco 0127) 5.0g
• NaCl 10.0g
• (Glucose 5.0g)

(Alternately use 25g of premix (LB Broth) containing all of the above ingredients and 5g glucose. )

1. Add all ingredients to a clean 2 liter flask that has been rinsed with deionized or
2. distilled water.
3. Add 1 liter of deionized or distilled water.
4. Add 0.5ml of 4N Sodium Hydroxide.
5. Stir to dissolve the dry ingredients using a magnetic stir bar and heat.
6. Aliquot the LB broth in the following manner:
   • 20 test tubes (20ml) containing 5ml LB Broth
   • 4 Erlenmeyer flasks (500ml) containing 100ml LB Broth.
   • 1 Spinner flasks (500ml working volume) containing 500ml LB Broth.
7. Cover test tubes and spinner flasks with plastic closures and the flasks with gauze covered cotton and aluminum foil.
8. Autoclave for 20min at 121 degrees Centigrade.
9. Store indefinitely at room temperature.
10. Add appropriate volume of sterile filtered Ampicillin to tubes or flasks prior to use.

1 Liter of Luria-Bertani (LB) Agar + Ampicillin

Weigh out:

• Tryptone (Difco 0123) 10.0g
• Yeast Extract (Difco 0127) 5.0g
• NaCl 10.0g
• Agar 15.0g

(Alternately use 25g of premix (LB Agar) containing all of the above ingredients.)

1. Add all ingredients to a clean 2 liter flask that has been rinsed with deionized or distilled water.
2. Add 1 liter of deionized or distilled water.
3. Add 0.5ml of 4N Sodium Hydroxide.
4. Heat while stirring using a magnetic stir bar until all dry ingredients have dissolved.
5. Plug with cotton wrapped in gauze.
6. Autoclave for 20min at 121 degrees Centigrade.
7. Sterilely add appropriate volume of sterile filtered Ampicillin to flask when agar flask is cool enough to hold.
8. Swirl flask to mix antibiotic with LB Agar.
9. Spread sterile petri dishes on previously disinfected lab bench (save plastic sleeves for later storage of petri dishes).
10. Pour agar plates and allow agar to solidify undisturbed, turn over to mark plates with the date and description of the media (LB/AMP).
11. Proof plates by incubating overnight at 37 degrees Centigrade; check for contamination, discarding any plates showing microbial growth.
12. Store proofed plates stacked in their original plastic sleeves for three months at 4 degrees Centigrade.