Team:Macquarie/Protocols/Making LB agar plates

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Revision as of 06:40, 7 August 2012

Media Preparation

• LB media:
  • Ingredients: Tryptone 10g, Yeast extract 5g, NaCl 10g, MilliQ water to 1000ml.
  • Methods: Dissolve 10g tryptone, 5g yeast extract and 10g NaCl in 800 mL MilliQ water, making use of a magnetic stirrer. Once dissolved, bring volume up to 1 liter using the MilliQ water. Autoclave 500 mL of the solution (121°C, 15 min, standard liquid cycle).

• LB agar:
  • Ingredients: LB media broth 1000ml, Bacto agar 15g.
  • Methods: Add the Bacto agar to the remaining 1000 mL of LB media and autoclave [121°C, 15 min, standard liquid cycle]. Add 250 µL of Chloroamphenicol, Ampicillin or Kanamyacin and mix well before plating out and setting agar. After cooling and antibiotic addition, the LB agar was plated out using aseptic technique. 14 LB agar plates were prepared and allowed to cool next to the Bunsen. After this all plates were aseptically sealed using parafilm and stored in the refrigerator.

• SOC media (for competent cells):
  • Ingredients: 2% w/v bacto-tryptone 20 g, 0.5% w/v bacto-yeast extract 1g, NaCl 400µl 5M, KCl250 µl 2M, 20mM MgSO4 0.4821 g,20mM glucose 0.7222g, MilliQ water to 200 mL.
  • Methods: The adjusted quantities were combined in 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, this was then adjusted to pH 7.0 with NaOH or HCl and brought up to 1L. The media was then sterilized by autoclaving.

• TB Buffer (for competent cells):
  • 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) are 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 4C.

• EDTA Buffer:
  • Ingredients:
  • Methods:

• TAE Buffer:
  • Ingredients:
  • Methods:

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

After autoclaving the LB agar plates were poured and each had ONE of the following: Chlor