Q1: The positive control (DHFR) is not synthesized.
1) Incubate the PUREfrex® reaction mixture by directly heating the reaction tube with heat block or water bath. Gas-phase chamber (e.g., incubator) takes time to warm the reaction mixture and the yield may be reduced.

2) Some of the components of the kit may lose their activity. Store the kit at a proper temperature to prevent loss of activity. Divide the reagents into aliquots to avoid the repeated freeze-thaw cycles as much as possible.

3) Cysteine may not have been added. In PUREfrex® 2.1, you can freely adjust the amount of reducing agents (Cysteine, DTT and GSH, etc.), but be sure to add Cysteine, which is a material of protein.
Q2: The protein of interest is not synthesized.
1)The template DNA may not contain the minimum required sequences. Template DNA for PUREfrex® must contain T7 promoter, ribosome binding site (SD sequence), start codon, and stop codon.

2) Too much or little DNA may be added to the reaction mixture. Regardless of plasmid DNA or PCR product, the template DNA should be added at a concentration of 0.5 to 3 ng per 1 kbp for 1 µL of the reaction mixture. Adding too much may reduce the protein yield also. The concentration of the template DNA added to the reaction mixture of PUREfrex® is defined as the number of DNA molecules (molar concentration) and it is added at a final concentration around 2 nM. About 2 nM is approximately 0.5 to 3 ng per 1 kbp for 1 µL of the reaction mixture. A 6 kbp plasmid DNA, for example, would be added at (0.5-3)×6 = 3-18 ng/µL, regardless of the length of the ORF.

3) The method for preparing the template DNA is inappropriate. Please see Tech Notes: "Preparation of the template DNA".

4) The template DNA contains a difficult-to-translate sequence. Confirm that the template DNA does not contain the sequences described in Tech Notes: "6 Tips about the template DNA Design".
Q3: The synthesized protein is insoluble.
1) Add molecular chaperones when synthesizing the protein. PUREfrex® contains no molecular chaperones. When the protein of interest is synthesized in the presence of molecular chaperones, it may become soluble. Additionally, better results may be obtained with PUREfrex® 1.0, in which the yield of the product is lower than PUREfrex® 2.0.

2) Synthesize the protein at a lower translation speed. Proteins generally fold more slowly than they are translated. Reducing the translation speed by lowering the reaction temperature from 37°C to 30°C or 25°C may increase the proportion of the target protein that is soluble. This will reduce the yield, so be sure to select conditions to efficiently produce the soluble protein.

Please see, "【Poster_MBSJ 2020】Investigation on how to synthesize active proteins by using a reconstituted cell-free protein synthesis system (PUREfrex®)"
Q4: The synthesized protein has no activity.
1) The synthesized protein may be insoluble. Check its solubility.

2) Factors required for proper activity (e.g., coenzymes, metal ions) may be missing. Since PUREfrex® is a reconstituted system, it contains no small molecules not relating to transcription and translation. Add the factors required for activity.

3) If the protein of interest requires correct disulfide bond formation, synthesize it in the presence of an additive to promote this formation (e.g. GSSG, DsbC). In that case, use PUREfrex® 2.1 in which reducing agent can be selected, because the efficiency of disulfide formation is affected by reducing agent.

Please see, "【Poster_MBSJ 2020】Investigation on how to synthesize active proteins by using a reconstituted cell-free protein synthesis system (PUREfrex®)"
Q5: The reaction mixture becomes turbid when sample buffer for SDS-PAGE is added.
The PUREfrex® reaction mixture has a relatively high salt concentration and may become turbid when heated after sample buffer containing SDS is directly added. To avoid turbidity, dilute the reaction mixture with at least equal volume of water before adding sample buffer. If this does not resolve the turbidity issue, heat the reaction mixture at a lower temperature (e.g., 37ºC) for a longer time (about 1 hour).