Q1: What is the point of template DNA preparation and optimization?
Please refer to the following page on the points for template DNA preparation and optimization.

Tech Notes: "Preparation of the template DNA"
Tech Notes: "6 Tips about the template DNA Design"
Q2: What sequences are required upstream of the gene for the protein of interest?
The template DNA must contain T7 promoter sequence and ribosome binding site (SD sequence) upstream of the gene encoding the protein of interest.
Q3: Can I use any promoter besides T7 promoter?
We recommend the template DNA with T7 promoter, because PUREfrex® contains T7 RNA polymerase for transcription. However, if you use other polymerases, please generate template DNA with the suitable promoter for the selected polymerase.
Q4: Is it possible to change the 5'UTR sequence?
However, we know that the 5'UTR sequence affects the protein synthesis; see our poster on the effect of 5'UTR sequences on yield. So far, the 5'UTR sequence we have designed has shown the highest yield. Each sequences in 5'UTR have different effects on the yield, so please adjust the sequence according to the purpose of your experiment.

Please see, "【Poster_MBSJ 2021】Exploration of the template DNA sequence suitable for the E. coli-based reconstituted cell-free protein synthesis system (PUREfrex®)".
Q5: Which stop codons can I use?
All of 3 stop codons (TAA, ochre; TAG, amber; TGA, opal) can be used with PUREfrex®, because it contains 2 release factors (translation termination factors) recognizing all 3 stop codons.
Q6: What sequences are required downstream of the gene for the protein of interest?
When you use circular DNA, T7 terminator sequence to terminate transcription must be placed downstream of the gene encoding the protein of interest. When you use linear DNA, add at least 10 nucleotides downstream from stop codon. T7 terminator sequence is not necessary when linear DNA is used.
Q7: When preparing the template DNA for PUREfrex by two-step PCR, are there any restrictions on the "arbitrary sequence of 10 bases or more" of the REV primer?
"REV primer" is basically flexible, but there are some points to be considered.

・The "taatga" after the C-terminus of the ORF means two stop codons.
・The length is more important than the sequence. If the length is shorter than 10 bases, the efficiency of protein synthesis will be affected, but if it is longer than 10 bases(like 20 bases or more), it will be fine.
・Avoid sequences that form a rigid secondary structure (ex, high GC content, etc.) just after the stop codon as much as possible.
・For example, a restriction enzyme site can be inserted in this region.
Q8: What amount of the template DNA is added to the reaction mixture?
0.5-3 ng per 1 kbp DNA for 1 µL of the reaction mixture will be fine.
The amount of DNA should be calculated based on number of DNA molecules. For example, in case of 6 kbp of plasmid, regardless of length of ORF, 3 to 18 ng of DNA should be added for 1 µL of the reaction mixture.
Q9: Can TE buffer be used for dissolving the template DNA?
Buffers containing EDTA should not be used for dissolving DNA, because EDTA inhibits transcription and translation reaction. We recommend dissolving the template DNA in EDTA-free buffer or Milli-Q water.
Q10: What are the points to use PCR product as the template DNA?
Although unpurified PCR reaction mixture can be directly used with PUREfrex®, do not add more than 10% (v/v) to the reaction mixture of PUREfrex®.

The efficiency of both transcription and translation reaction may decrease when the salt concentration is altered by carryover from the PCR mixture.

If the concentration of the PCR product is low, prepare DNA solution with a sufficient concentration using a DNA purification kit instead of adding more unpurified PCR mixture to PUREfrex®.

Please see, Tech Notes: "Preparation of the template DNA"
Q11: What purity do I need for my template PCR product?
PCR product should be a single band on an electrophoresis gel. If you found unexpected bands, modify the PCR condition to reduce byproducts.

The purity of the PCR product affect the efficiency of protein synthesis because proteins can be also synthesized from PCR-byproducts.

If byproducts are unable to eliminate by changing the PCR conditions, excise the target band from the gel and purify the DNA. Excise the band from the gel without using ultraviolet light, which can damage DNA (i.e., impair transcription). Blue light may be used, but exposure time should be kept to a minimum.

Please see, Tech Notes: "Preparation of the template DNA"
Q12: Can synthetic DNA be used as a template?
Synthetic DNA fragments containing promoter sequences can be used as template DNA. In addition, template DNA can be prepared from synthetic DNA of ORFs by PCR using primers that include the promoter sequence and other necessary sequences.

However, depending on the manufacturer of the gene synthesis, the delivered product may contain RNase, which inhibits the protein synthesis reaction. If the protein of interest cannot be synthesized, try adding an RNase inhibitor or inactivating the RNase.

Also, if the product is delivered as a plasmid, please check the notes for using the plasmid as a template DNA (Tech Notes: "Preparation of the template DNA").
Q13: What types of plasmid vectors can be used as template DNA?
Vectors that contain T7 promoter, SD sequence, and T7 terminator can be used such as pET vectors (Merck) and pQE vectors (Qiagen). The presence of the lac operator sequence may decrease the protein yield. We recommend using vectors without the lac operator sequence (e.g., pET17).
Q14: What are the points of plasmid DNA preparation?
In preparing plasmid DNA, make sure that the RNase used for purification is not contaminated in the final purified DNA. Using a filter-based purification kit such as QIAprep Spin Miniprep Kit (Qiagen) or Wizard Plus SV Minipreps DNA Purification System (Promega), for example, results in contamination of RNase A (contained in Lysis buffer) in to the final purified DNA solution.

If you add this solution to the PUREfrex® reaction mixture as template DNA without further treatment, transcription products and other RNAs in the reaction mixture are digested, which inhibit protein synthesis.

Therefore, treat the DNA with phenol/chloroform to denature RNase and then re-purify the DNA by ethanol precipitation to prepare it with free of RNase activity. Adding RNase inhibitor to the PUREfrex® reaction mixture is also effective.

Plasmid DNAs purified with Plasmid Mini Kit (Qiagen) has minimal contamination with RNase because the plasmid DNA eluted from resin is precipitated by adding isopropanol. Plasmid DNA purified with this kit are verified to use without any treatment.

Please see, Tech Notes: "Preparation of the template DNA"
Q15: How to use RNA as a template?
In case of using mRNA to synthesize the protein of interest, make sure that the mRNA contains SD sequence upstream of start codon. Add mRNA usually at a concentration of 0.1 to 1 µM to the reaction mixture. Since the optimal concentration depend on the sequence and purity of the mRNA, we recommend examining the optimal concentration in reference to this concentration range.