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”.