The template DNA should have the structure of T7 promoter, RBS, His-tag and T7 terminator as shown below. Depending on where the His-tag is attached, you can choose between two different types of expression, folding, and separation purification. At this time, the template type can be either plasmid DNA or PCR product. When preparing with a PCR product, it is advantageous to prepare several types of templates quickly, but the yield of protein synthesis may be slightly lower than that of plasmid DNA. Especially, when using SECF method for long time reaction for large-scale expression, the stability of template is important, so we recommend plasmid template.
1) When plasmid DNA is used as a template, It is recommended to use pBIVT vector (Cat. No. K-7350, Bioneer) which is exclusive to in vitro translation, but T7 system, RBS, and His-tag for purification can also be used. Using the AccuPrep® Nano-Plus Plasmid Mini Extraction Kit (Cat. No. K-3112, Bioneer), cloned plasmid DNA can be isolated with high-yield, high-purity only within 10 minutes
2) When using a PCR product as a template, ExiProgen™ ProXpress PCR Template Kit (Cat. No. K-7400, Bioneer) can be used for amplification of the target gene for preparing large quantities of the templates by PCR without the cloning step. For the best results, the purity of the acquired template DNA should be in the range of A260 / 280 ratio of 1.8 - 2.0.