Q1: 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.
The amount of DNA is 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 is added for 1 µL of the reaction mixture.
Q2: 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.
Q3: What are key points to use PCR product as the template DNA?
We recommend that the template DNA is prepared by two-step PCR.
Overview of two-step PCR is illustrated in Fig. 5 and primer sequence for two-step PCR is shown in Table 2.
Text sequence is also available.
Purity and Amount of PCR product
PCR product should be a single band on an electrophoresis gel. Otherwise unexpected polypeptides may be synthesized. If there are still extra bands in spite of optimizing PCR condition, please purify the desired band by gel extraction.
We recommend blue light for gel extraction, not UV light. UV light causes DNA damage, which can lead to an unexpected termination of transcription. You should cut the gel quickly even using blue light.
PCR reaction mixture containing the amplified template DNA can be directly added to PUREfrex® reaction mixture. However, some components in the PCR reaction mixture inhibit transcription and/or translation reaction.
To prevent carrying inhibitory components from the PCR reaction mixture, we recommend adding less than 0.1 volume of the PCR reaction mixture to PUREfrex® reaction mixture. If the concentration of the template DNA is not enough for protein synthesis, you may concentrate DNA solution with a purification kit or by gel extraction.
Q4: What are key points to use plasmid DNA as the template DNA?
To include essential elements
T7 promotor, SD sequence, and T7 terminator are essential elements. We don’t recommend using vectors including Lac operator because it causes decrease on productivity depending on the target protein.
RNase digests the transcribed RNA in the reaction mixture, resulting reduction of the productivity of the target protein. Many commercial kits can be used for purifying plasmid DNA. However, in some cases, RNase in the lysis buffer is brought over to the purified DNA fraction.
If there is a possibility that RNase is contained in the purified plasmid DNA fraction, we recommend Phenol/Chloroform extraction followed by ethanol or isopropanol precipitation for removing RNase.
Addition of RNase inhibitor to the PUREfrex® reaction mixture is also effective.
Q5: Do I need optimization of the sequence of template DNA for PUREfrex®?
We recommend that you use the optimal codons for E. coli because PUREfrex® utilizes translation system derived from E. coli. Moreover, N-terminal codons (2nd-6th codon following 1st ATG) should be changed to AT-rich codon (not rather than the most frequently used codon) to increase the productivity.
Please refer the results in our posters, which are partially described in Japanese but all results in English.
“Improvement of translational efficiency by N-terminal codon optimization in the reconstituted cell-free protein synthesis system” (2016)
»Poster1; Fab, His tag, etc. (0.7 MB)
»Poster2; various proteins, membrane protein, etc. (2.5 MB)