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

Figure 1. Preparation of LB agar plates.

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^oC, 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^oC, 15 min, standard liquid cycle).