Difference between revisions of "Team:Bielefeld-CeBiTec/Media"

m
Line 225: Line 225:
 
                   <ul><li> Stock solutions for CFPS</li>
 
                   <ul><li> Stock solutions for CFPS</li>
 
<ul>
 
<ul>
−
     <li> 1.5 M Spermidine and 1 M putrescine solutions are prepared according to supporting material from <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Yang2012a">Yang et al. 2012a</a>. Briefly, a total of 23 mL of autoclaved MilliQ water is added in small steps to a 5 g bottle of spermidine. After each step, the bottle is mixed vigorously and the liquid is poured into a beaker where it is continously stirred. After rinsing out the whole bottle, the homogenous solution is aliquoted, flash freezed and stored at -80 °C. The procedure is similar for putrescine, though volumes differ.
+
     <li> 1.5 M Spermidine and 1 M putrescine solutions are prepared according to supporting material from <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Yang2012a">Yang et al. 2012a</a>. Briefly, a total of 4.59 mL of autoclaved MilliQ water is added in small steps to a 1 g bottle of spermidine. After each step, the bottle is mixed vigorously and the liquid is poured into a beaker where it is continously stirred. After rinsing out the whole bottle, the homogenous solution is aliquoted, flash freezed and stored at -80 °C. The procedure is similar for putrescine, though volumes differ.
 
     </li>
 
     </li>
 
      
 
      

Revision as of 08:47, 15 August 2015

iGEM Bielefeld 2015


Media

LB

  • For 1 liter LB:
    • 20 g LB powder
    • Fill the bottle with deionized H2O

  • For 1 liter LB-plates:
    • 18 g LB powder
    • 16 g Select Agar
    • Fill the bottle with deionized H2O

SOC

  • Add the following components for 900 ml of distilled H2O:
    • 20 g Trypton
    • 5 g Bacto Yeast Extract
    • 2 ml of 5 M NaCl
    • 2.5 ml of 1 M KCl
    • 10 ml of 1 M MgCl2
    • 10 ml of 1 M MgSO4
    • 20 ml of 1 M glucose

Buffers

50X TAE Stock Solution

  • For each litre of solution:
    • 242 g Tris Base (MW=121.1)
    • 57.1 ml Glacial Acetic Acid
    • 100 ml 0.5 M EDTA

  • mix Tris with stir bar to dissolve in about 600 ml of ddH2O.
  • add the EDTA and Acetic Acid.
  • bring final volume to 1 l with ddH20.
  • store at room temperature.

  • Note: Final (1x) working concentration:
    • 0.04 M Tris - Acetate
    • 0.001 M EDTA

Lysis and binding buffer for purification of lacI

  • For each 200 ml of solution:
    • 5.844 g NaCl
    • 0.272 g imidazole
    • 5.8 ml of 86 % glycerol
    • 0.406 g MgCl2
    • 0.2 g lysozyme

  • Add all these ingredients to a 50 mM sodium phosphate buffer (pH 8.0). Prior to use add DTT to a final concentration of 1mM and tween 20 to a final concentration of 0.1 %.
  • store at room temperature.

  • Note: Final (1x) working concentration:
    • 50 mM Sodiumphosphate
    • 500 mM NaCl
    • 20 mM imidazole
    • 2.5 % glxcerol
    • 1 mM DTT
    • 10 mM MgCl2
    • 0.1 % tween 20
    • 1 mg/ml lysozyme

Equilibration buffer for lacI purification

  • For each litre of solution:
    • 29.22g NaCl
    • 1.36 g imidazole
    • 29 ml of 86 % glycerol

  • Add all these ingredients to a 50 mM sodium phosphate buffer (pH 8.0). Prior to use add DTT to a final concentration of 1mM.
  • store at room temperature.

  • Note: Final (1x) working concentration:
    • 50 mM sodium phosphate
    • 500 mM NaCl
    • 20 mM imidazole
    • 2.5 % glycerol
    • 1 mM DTT

Elution buffer for purification of lacI

  • For each litre of solution:
    • 17.53 g NaCl
    • 13.6 g imidazole

  • Add all these ingredients to a 20 mM sodium phosphate buffer (pH 7.4).
  • store at room temperature.

  • Note: Final (1x) working concentration:
    • 20 mM sodium phosphate
    • 300 mM NaCl
    • 200 mM imidazole

Buffer for dialysis of lacI

  • For each litre of solution:
    • 50 g glucose

  • Add the glucose to a 200 mM potassium phosphate buffer (pH 7.6). Prior to usage add DTT to a final concentration of 1 mM
  • store at room temperature.

  • Note: Final (1x) working concentration:
    • 200 mM potassium phosphate
    • 5 % glucose
    • 1 mM DTT

Buffers for CFPS

  • Stock solutions for CFPS
    • 1.5 M Spermidine and 1 M putrescine solutions are prepared according to supporting material from Yang et al. 2012a. Briefly, a total of 4.59 mL of autoclaved MilliQ water is added in small steps to a 1 g bottle of spermidine. After each step, the bottle is mixed vigorously and the liquid is poured into a beaker where it is continously stirred. After rinsing out the whole bottle, the homogenous solution is aliquoted, flash freezed and stored at -80 °C. The procedure is similar for putrescine, though volumes differ.