Team:Macquarie Australia/Protocols/Making LB agar plates



  • LB media: (performed 3 times to make 3L total)
    • Ingredients: Tryptone 10g, Yeast extract 5g, NaCl 10g, MilliQ water to 1000ml.
    • Methods: Dissolved 10g tryptone, 5g yeast extract and 10g NaCl in 800 mL MilliQ water, making use of a magnetic stirrer. Once dissolved, brought volume up to 1L using MilliQ water. Autoclaved 1000ml of the solution (121°C, 15 min, standard liquid cycle).

  • LB agar: (performed 3 times to make 3L total)
    • Ingredients: LB media broth 1000ml, Bacto agar 15g.
    • Methods: Added the 15 Bacto agar to 1000 mL of LB media and autoclave [121°C, 15 min, standard liquid cycle]. Add 1000µl of Chloroamphenicol (25 mg/mL), Ampicillin (50 mg/mL) or Kanamycin (30 mg/mL) and mixed well before plating out and setting agar. After cooling and antibiotic addition, the LB agar was plated out using aseptic technique. In total, 31 Ampicillin LB agar plates, 33 Chloramphenicol LB agar plates & 32 Kanamycin LB Agar plates resulted. All plates were aseptically sealed using parafilm and stored in a refrigerator. Ultimately, this procedure resulted in three 1L LB agar solutions with an different antibiotic in each.

LB ready for the autoclave
Pouring of LB agar plates
Ready for parafilm

SOC media (for competent cells): Ingredients: 10g bacto-tryptone, 2.5g bacto-yeast, 1000µl 5M NaCl, 417 µL 3M KCl, 1.205g 20mM MgS04, 805g 20 mM D-glucose & 500 mL Milli-Q water.
Methods: The adjusted quantities were combined in a 1L measuring column with constant stirring and then placed in the autoclave for sterilization.

SOB Media (for competent cells):
Ingredients: Bacto Tryptone 20g, Bacto Yeast 5g, NaCl 0.59, KCl 0.19g, 2.03g of MgCl2 (10mL of 1M), 1.2 g of MgS04 and MilliQ water to 900ml.
Methods: Ingredients were combined and was then adjusted to pH 7.0 with NaOH or HCl and brought up to 1L. The media was then sterilized by autoclave.

TB Buffer
Ingredients: 3g of PIPES, 10.9 grams of MnCl2-4H20, 2 grams of CaCl2-2H20, 18.6 grams of KCl
Methods: All components (except for MnCl2-4H20) were mixed and dissolved in 500 ml of water and pH adjusted to 6.7 with KOH. Then, MnCl2-4H20, is dissolved in 300 ml of water, mixed and solution adjusted to 1L. Sterilization via filtration followed through a pre-rinsed 0.45um filter unit and stored at 4°C.

EDTA Buffer:
Ingredients: 37.22g of EDTA solid, 180 mL of water and pH adjusted to 8.0 using 10 M NaOH.
Methods: Components were combined then pH adjusted.

TAE Buffer:
Ingredients: 121.2g of Tris base (dissolved in water) with 28.55 mL of glacial acetic acid & 50 mL 0.5 M EDTA (pH 8.0)
Methods: A total volume of 500 mL was made up as a 50x stock solution using all components.

References: SOB media & TB buffer modified from Inoue et al., 1992 (Competent Cell Method)