Team:Macquarie Australia/WetLab/Protocols/Media

Media & Plates

Well prepared media are crucial to successful selection and growth of organisms.

LB AGAR PLATE PREPARATION

• Add 15g Bacto agar to 1000mL of LB media and autoclave (121oC for 15mins)
• 2. Add 1000uL of Chloroamphenicol (25mg/mL), Ampicillin (50mg/mL) or Kanamycin (30mg/mL) and mix well before plating out and setting the agar.

LB MEDIA

Ingredients: Tryptone 10g, Yeast extract 5g, NaCl 10g, Milli-Q water to 1000mL Methods: Dissolved 10g tryptone, 5g yeast extract and 10g NaCl in 800mL Milli-Q water, making use of a magnetic stirrer. Once dissolved, bring volume up to 1 L using Milli-Q water. Autoclave 1L of the solution (121°C, 15 min, standard liquid cycle).

SOC MEDIA (FOR COMPETENT CELLS)

Ingredients: 10g bacto-tryptone, 2.5g bacto-yeast, 1000µl 5M NaCl, 417µL 3M KCl, 1.205g 20mM Mg2SO4, 805g 20mM D-glucose & 500mL 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 (121°C, 15 min, standard liquid cycle).

TB BUFFER

Ingredients: 3g PIPES, 10.9g 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: 37.22g 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: 121.2g Tris base (dissolved in water) with 28.55mL of glacial acetic acid & 50mL 0.5M EDTA (pH 8.0) Methods: A total volume of 500 mL was made up as a 50x stock solution using all components.