Team:Macquarie/Protocols/Making LB agar plates

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Revision as of 08:24, 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 1000µl of Chloroamphenicol (25 mg/mL), Ampicillin (50 mg/mL) or Kanamyacin (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. *** how many plates were done*** All plates were aseptically sealed using parafilm and stored in a 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 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 (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 4°C.

• EDTA Buffer:
  • Ingredients: 18.61g of EDTA solid, 90 mL of water and pH adjusted to 8.0 using 10 M NaOH.
  • Methods: Components combined then pH adjusted.

• TAE Buffer:
  • Ingredients: 121g 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)

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