Team:Macquarie/Protocols/Making LB agar plates

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Revision as of 08:44, 7 August 2012

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 1000ml 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 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 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.

LB ready for the autoclave

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 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)