Table 2. In vitro protein synthesis using cell-free system based on S30-T7 lysate.

A. Materials and solutions required:	Notes:
1M HEPES-KOH (pH = 8)
1M magnesium acetate
1M potassium acetate
5.2 M ammonium acetate
50% (v/v) Polyethylene glycol 6000 (PEG)
0.5 M 3-phosphoglycerate (3-PGA)
50 mM of 17 amino acids	These 17 amino acids are alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, threonine, and valine.
50 mM of 3 amino acids	These 3 amino acids are tryptophan, phenylalanine, and tyrosine.
100 mM Adenine triphosphate (ATP)
50 mM Guanidine triphosphate (GTP)
100 mM Uridine triphosphate (UTP)
S30-T7 lysate	Prepared according to Table 1
DNase, RNase free H2O
B. Equipment:	Notes:
Floor incubator shaker or a Thermomixer®
C. Procedure:
1.1Prepare the CFPS reaction mixtures according to Table 3. Make sure to thaw and add the S30-T7 lystae just prior to the incubation and protien production step. 1.2Incubate the reaction using a floor incubator shaker at 250 rpm, or a Thermomixer® at 1200 rpm at a constant temperature for 2 hours. When this protocol is first used, it is recommened to incubate the reaction at 37°C. In addition, perform a parallel reaction without DNA, to obtain a negative control. 1.3Evaluate the produced protein amount using a suitable method, according to the target protein properties.