Team:Bielefeld-CeBiTec/Media

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

Buffers

• S30A buffer is prepared according to Sun et al. 2013 and contains
  • 14 mM Mg-glutamate
  • 60 mM K-glutamate
  • 50 mM TRIS
  at pH = 7.7 (with acetic acid), autoclaved, stored at 4 °C. Right before use, DTT is added to 2 mM final concentration.
• S30 buffer (buffer A from Kwon and Jewett 2015) with lower TRIS concentration
  • 14 mM Mg-glutamate
  • 60 mM K-glutamate
  • 10 mM TRIS
  at pH = 8.2 (with acetic acid), autoclaved, stored at 4 °C. Right before use, DTT is added to 2 mM final concentration.

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.
• NTP mixture is prepared from ATP, CTP, GTP and UTP stock solutions (100 mM) from Thermo Scientific. 20x NTP premix contains 30 mM of ATP and GTP, 18 mM CTP and UTP.
• 330 mM Phosphoenolpyruvate (PEP) stock solution is prepared and pH is adjusted with 5 M KOH according to supporting material from Yang et al. 2012a