Team:Macquarie/Protocols/Making LB agar plates

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Revision as of 05:38, 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 5g, Bacto Yeast 1.25g, NaCl 0.124g, KCl 0.0475g MilliQ water to 900ml.
- Methods: Ingredients were combined, this was then adjusted to pH 7.0 with NaOH or HCl and then to 250mL with MilliQ water. The media was then sterilized by autoclaving.

@@ Line 2: / Line 2: @@
-*'''LB media Method:'''
+*'''LB media:'''
-**'''Ingredients:'''-Tryptone 10g, Yeast extract 5g, NaCl 10g, MilliQ water to 1000ml.
+**'''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).
+**'''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 Method:''' Add 7.5 g Bacto agar to the remaining 500 mL of LB media and autoclave [121°C, 15 min, standard liquid cycle]. Add 250 µL of Chloroamphenicol 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.
+*'''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):''' A mixture of 4g 2% w/v bacto-tryptone (20 g), 1g 0.5% w/v bacto-yeast extract, 400µl 5M NaCl, 250 µl 2M KCl, 20mM MgSO4 (0.4821 g), 0.7222g 20mM glucose and MilliQ H2O to 200 mL. The adjusted quantities were combined in 1l measuring column with constant stirring and then placed in the autoclave for sterilization.
+*'''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):''' Distilled H2O of volume 900ml was added with 5g Bacto Tryptone, 1.25g Bacto Yeast Extract, 0.124 grams NaCl and 0.0475 grams KCl. This was then adjust to pH 7.0 with NaOH or HCl and then to 250mL with distilled H2O. The system was then sterilized by autoclaving.
+*'''SOB Media (for competent cells):'''
+**'''Ingredients:''' Bacto Tryptone 5g, Bacto Yeast 1.25g, NaCl 0.124g, KCl 0.0475g MilliQ water to 900ml.
+**'''Methods:''' Ingredients were combined, this was then adjusted to pH 7.0 with NaOH or HCl and then to 250mL with MilliQ water. The media was then sterilized by autoclaving.