• 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

• 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

• 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

• 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

• 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

• 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

• 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

• 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.