Team:Macquarie/Protocols/Making LB agar plates

From 2012.igem.org

(Difference between revisions)
(→Media Preparation)
(→Media Preparation)
Line 1: Line 1:
-
==='''Media Preparation'''===
+
==='''Preparation'''===
*'''LB media:'''  
*'''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 1000ml of the solution (121°C, 15 min, standard liquid cycle).
*'''LB agar:'''  
*'''LB agar:'''  
**'''Ingredients:''' LB media broth 1000ml, Bacto agar 15g.
**'''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.  
+
**'''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 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.  
Line 20: Line 20:
**'''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.  
**'''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.
**'''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):'''
*'''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
**'''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.
**'''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:'''
*'''EDTA Buffer:'''
**'''Ingredients:''' 18.61g of EDTA solid, 90 mL of water and pH adjusted to 8.0 using 10 M NaOH.  
**'''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.  
**'''Methods:''' Components combined then pH adjusted.  
 +
*'''TAE Buffer:'''
*'''TAE Buffer:'''
Line 33: Line 36:
**'''Methods:''' A total volume of 500 mL was made up as a 50x stock solution using all components.  
**'''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)
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
After autoclaving the LB agar plates were poured and each had ONE of the following: Chlor

Revision as of 08:26, 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 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

Retrieved from "http://2012.igem.org/Team:Macquarie/Protocols/Making_LB_agar_plates"