A1:
　You can use plasmid DNA that expresses protein under the control of the general T7 promoter, which consists of T7 promoter, SD sequence, N-terminal tag sequence(optional but recommended), target protein gene, and T7 terminator. The plasmid can also be prepared from the control plasmid CAT provided in the kit by replacing the CAT gene with an appropriate N-terminal tag sequence and the target protein gene. The N-terminal sequence of the protein coding sequence to be synthesized greatly affects the yield of the protein. It is recommended to add an N-terminal tag sequence (Ref.) that increases the protein yield. Widely used N-terminus His x 6 tag often reduce the yield significantly, so we recommend to replace it with the tags in the Ref.

A2:
　After purification with a commercially available plasmid purification kit, we recommend additional purification by (1) PCR product purification kit or (2) phenol/chloroform extraction followed by ethanol precipitation. In addition, it is recommended to check the purity and approximate concentration of the plasmid by agarose electrophoresis.

　Plasmids purified with commercial plasmid purification kits often contain residual RNaseA in the final purified plasmid solution. If used as is, RNaseA may inactivate the cell-free protein synthesis system, resulting in the total inability to synthesize proteins. Even when the same plasmid purification kit is used, the amount of RNaseA residue can vary greatly due to differences in detailed procedures. For stable protein synthesis, additional purification is strongly recommended to ensure the removal of RNaseA. Phenol chloroform extraction followed by ethanol precipitation is the most reliable method for additional purification, but commercially available PCR product purification kits (DNA Clean & Concentrator-5 by Zyme Research, etc.) can also be used.
　We have experience that plasmids purified with NucleoBond® Extra Midi (Takara/Machrei Nagel) can be used for protein synthesis without additional purification.
　When the concentration of a purified plasmid sample is estimated by UV absorption (A₂₆₀), the concentration of the plasmid may be overestimated due to absorption caused by impurities in the solution (genomic DNA or small molecules). It is recommended that the concentration should be validated and the quality of the plasmid should be checked by agarose electrophoresis.

NucleoBond is a registered trademark of Machrei Nagel GmbH.

A3:
　Design a linear dsDNA sequence from the T7 promotor to the T7 terminator of the plasmid template, with additional a few dozen or more bases to upstream of the T7 promoter and downstream of the T7 terminator. Two-step PCR method (Ref.) is also recommended.

　Cell-free protein synthesis reaction mixture has some exonuclease activity, so adding extra sequences upstream of the T7 promoter and downstream of the T7 terminator will increase the exonuclease resistance of the linear template DNA and stabilize the protein synthesis.

A4:
　PCR product without any purification can be directly used as a template of cell-free protein synthesis. Usually, the amount of template DNA required for protein synthesis is about one tenth volume of the protein synthesis reaction mixture. Thus, the final concentration of the template DNA is about 10 to 30 ng/µL.
　Please start the protein synthesis reaction (incubation) immediately after addition of linear templates into the reaction solution. If starting the synthesis reaction is delayed, the linear template may be degraded before launch of protein synthesis and the amount of protein synthesized may decrease.

A5:
　 Please cool the reaction solution on ice for 5 minutes, allow it to precipitate sufficiently. Then, centrifuge the reaction solution and collect the supernatant. For His tag affinity purification of proteins, dilute the reaction solution 5-fold or more with an appropriate buffer and then apply to affinity resin.

　The reaction solution after synthesis contains inorganic phosphate salts, which may clog the column. By cooling the reaction solution for a while after synthesis, inorganic phosphate salts can be precipitated out and can be removed by centrifugation.
　The reaction solution contains a considerable amount of amine compounds such as amino acids that may inhibit His tag binding onto the affinity resin. Reducing the concentration of amine compounds enables His tag binding onto the resin.

A6:
　The reaction solutions of the Musaibo Kun Quick, SI, SI SS, and Start kits contain PEG, which distorts the SDS-PAGE image of low-molecular-weight. Please remove PEG by acetone precipitation (the procedure is described in the kit manual) before preparing SDS-PAGE samples. The reaction solution of Musaibo Kun SI (PEG-free), SI SS (PEG-free) and N series kits does not contain PEG, so you can prepare the SDS-PAGE samples by a standard procedure without acetone precipitation.

A7:

Causes	Recommended measures
Reduced kit performance	(1) Please confirm that the product is within the expiration date indicated on the product label. (2) Please confirm that the product has been stored at an appropriate temperature. (3) If the product has been stored properly within the expiration date, please contact us.
Inappropriate template DNA	(1) Please check if the template DNA sequence is appropriate. (2) Please check if the concentration is appropriate by agarose gel electrophoresis. (3) When using plasmid DNA prepared with a commercially available plasmid purification kit, please perform additional purification by phenol/chloroform extraction followed by ethanol precipitation to remove a trace amount of residual nucleases. (4) Please try to confirm the expression using Musaibo Kun N100 or Start kit, that has a small scaled high-yield dialysis mode configuration.
Problems due to the nature of the protein	Some unstable proteins tend to degrade or precipitate during the protein synthesis reaction. (1) Lowering reaction temperature may reduce degradation or improve solubility although yield is decreased. Please try incubation at 25°C (2) When kits other than Quick or N mini are used, shortening reaction time may reduce degradation or improve solubility although yield is decreased. Please try 4 hour or less of reaction time. (3) Insertion of a tag sequence that improves solubility may change solubility of whole protein. The amount of expression varies greatly depending on the amino acid sequence especially at N-terminal region. (1) Please add or change N-terminal tag. (2) According to the protein specification, please try optimizing the construct by deletion, extension or mutilation.