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Team:Macquarie Australia/Making LB Agar Plates
From 2013.igem.org
Preparation
- LB media: (performed 3 times to make 3 L total)
- Ingredients: Tryptone 10 g, Yeast extract 5 g, NaCl 10 g, Milli-Q water to 1000 mL.
- Methods: Dissolved 10 g tryptone, 5 g yeast extract and 10 g NaCl in 800 mL Milli-Q water, making use of a magnetic stirrer. Once dissolved, brought volume up to 1 L using Milli-Q water. Autoclaved 1000 mL of the solution (121°C, 15 min, standard liquid cycle).
- LB agar: (performed 3 times to make 3 L total)
- Ingredients: LB media broth 1000 mL, Bacto agar 15 g.
- Methods: Added the 15 g Bacto agar to 1000 mL of LB media and autoclave [121°C, 15 min, standard liquid cycle]. Add 1000 µl 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 1 L LB agar solutions with an different antibiotic in each.
SOC media (for competent cells):
Ingredients: 10 g bacto-tryptone, 2.5 g bacto-yeast, 1000 µl 5M NaCl, 417 µL 3M KCl, 1.205 g 20mM MgSO4, 805 g 20mM D-glucose & 500 mL Milli-Q water.
Methods: The adjusted quantities were combined in a 1 L measuring column with constant stirring and then placed in the autoclave for sterilisation.
SOB Media (for competent cells):
Ingredients: Bacto Tryptone 20 g, Bacto Yeast 5 g, NaCl 0.59 g, KCl 0.19 g, 2.03 g MgCl2 (10 mL of 1M), MgSO4 1.2 g and Milli-Q water to 900 mL.
Methods: Ingredients were combined and was then adjusted to pH 7.0 with NaOH or HCl and brought up to 1 L. The media was then sterilised by autoclave.
TB Buffer
Ingredients: 3 g PIPES, 10.9 g MnCl2-4H2O, 2.0 g CaCl2-2H2O, 18.6 g KCl
Methods: All components (except for MnCl2-4H2O) were mixed and dissolved in 500 mL of water and pH adjusted to 6.7 with KOH. Then, MnCl2-4H2O, was dissolved in 300 mL of water, mixed and solution adjusted to 1 L. Sterilisation via filtration followed through a pre-rinsed 0.45 µm filter unit and stored at 4°C.
EDTA Buffer:
Ingredients: 37.22 g EDTA solid, 180 mL of water and pH adjusted to 8.0 using 10M NaOH.
Methods: Components were combined then pH adjusted.
TAE Buffer:
Ingredients: 121.2 g Tris base (dissolved in water) with 28.55 mL of glacial acetic acid & 50 mL 0.5M 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)
