RNase contamination Commercial mini-prep kits are a useful tool for the preparation of template DNA but are often the source of introducing RNase A to the in vitro protein synthesis reaction. The inclusion of RNase Inhibitor (NEB #M0314S, 20 units/25 μl reaction) often overcomes this problem. Template DNA design is compromised Ensure that the sequence of the template DNA is correct. The coding region as well as the regulatory sequences need to be correct and in-frame to ensure that translation is initiated properly and that a full-length product is made. Non-optimal regulatory sequences and/or spacing may adversely affect translational efficiency. Translation initiation is a key step for successful protein synthesis. Secondary structure or rare codons at the beginning of the mRNA may compromise the initiation process and adversely affect protein synthesis. The addition of a good initiation region (e.g. first ten codons of maltose binding protein) may help, assuming that adding residues to the target sequence can be tolerated. Alternatively, using PCR to modify the 5’ end of the target gene can be a successful strategy to eliminate secondary structural elements or rare codons. Template DNA is contaminated Inhibitors of transcription or translation may be present in the DNA. A simple mixing experiment (control DNA + target DNA, compared to control DNA alone) will reveal whether inhibitors are present. Inhibitors in the target DNA will reduce the yield of the control protein. Do not use DNA purified from agarose gels as they often contain inhibitors of translation (e.g. ethidium bromide). Residual SDS from plasmid preparation protocols is another common contaminant and can be removed by phenol:chloroform extraction and ethanol precipitation. When performing ethanol preciptation we recommend the use of sodium acetate rather than ammonium acetate, a known inhibitor of translation. Be careful to remove all traces of ethanol. Templates produced by PCR need to be free of non-specific amplification products. These contaminants may contain transcription signals and thus compete for and titrate out transcription and/or translation components. As a result, yields may suffer and unwanted truncated products may be produced. Template DNA concentration is not optimal The concentration of template DNA is important as in vitro protein synthesis is a balance of transcription and translation. Too little template reduces the amount of actively translated mRNA while too much template results in the overproduction of mRNA and the overwhelming of the translational apparatus. We recommend 250 ng of template DNA for a 25 μl reaction. Optimization with different amounts of template DNA (e.g. 25-1000 ng) may improve yield of a particular target protein. If UV absorbance was used to calculate the concentration of the template DNA, be aware that RNA or chromosomal DNA will also absorb UV light. If your sample has significant amounts of RNA or chromosomal DNA, the actual amount of template DNA may be lower than the calculated amount. The 260 nm/280 nm ratio should be 1.8. Running some of the template DNA on an agarose gel may reveal the presence of other nucleic acids as well as any degradation or incorrect size of the template DNA.