All myTXTL kits support plasmid or linear DNA templates as well as mRNA.

myTXTL supports all promoters used in E. coli protein expression, including promoters that rely on the endogenous E. coli transcription machinery and those that require a separate RNA polymerase such as T7. Inducible plasmids require that the inducer be added to obtain the highest protein yield. pET (T7lac promoter) systems need 1 mM IPTG, for example, otherwise follow the guidance in the following table for inducer and plasmid concentrations in the myTXTL reaction.

We recommend the following inducer and plasmid concentrations when using some common inducible promoters:

PromoterInducerRecommended Inducer Concentration in myTXTLUnitsRecommended Plasmid Template Concentration in myTXTLUnits
T7lacIPTG1mM10nM
TetAaTc20µg/mL20nM
araBADL-Arabinose2%20nM

It may be necessary at times to add components to the myTXTL system from sources that contain material of unknown effect in the myTXTL system, the following is a guide for what chemicals/reagents may be tolerated without a loss in performance:

1) Glycerol is tolerated up to 0.1% of myTXTL reaction volume

2) DMSO is tolerated up to 1% of myTXTL reaction volume

3) EDTA is tolerated up to 0.1 mM of myTXTL reaction volume

4) Tris-HCl (pH 8) is tolerated up to 50 mM of myTXTL reaction volume

5) CaCl2 is tolerated up to 1 mM of myTXTL reaction volume

6) MgCl2 is tolerated up to 1 mM of myTXTL reaction volume

7) NaCl is tolerated up to 50 mM of myTXTL reaction volume

Yes, it is expected that protein yield resulting from linear templates is diminished compared to its circular plasmid version. A decrease of 10-30% is considered to be within the normal range for the Pro kit. This decrease tends to be minimal in the Antibody/DS Kit.

Yes, it is expected that protein yield resulting from linear templates is diminished compared to its circular plasmid version. A decrease of 10-30% is considered to be within the normal range.

myTXTL requires high quality template DNA, which should be free of nucleases (DNases, RNases) and inhibitors of the TXTL machinery (e.g. EDTA, ethidium bromide, SDS, Cl- ions, ethanol). Preparation of plasmid DNA with standard commercial kits usually involves sample treatment with RNase, which may not be completely removed during downstream processing. Thus, we strongly recommend subjecting the prepared DNA to either a commercial PCR clean-up kit or standard phenol-chloroform extraction and ethanol precipitation. Ideally, template DNA is in deionized, nuclease-free water. Please note, introducing Mg2+and K+ ions can compromise the kit performance, as they are extremely critical for transcription and translation, and are optimized in the master mix. Plasmid DNA prepared with a ZymoPure system purification kit or obtained from a commercial vendor can generally skip this extra purification step. Linear templates generated via PCR require just a single PCR Purification kit clean up (or magnetic bead cleanup) step or no clean-up if the PCR reaction is being diluted directly into the myTXTL reaction (be sure glycerol in the reaction is 0.1% or lower). See kit manual for additional recommendations.

Apart from standard biochemical methods like Coomassie-stained SDS-PAGE and western blot analysis, the great advantage of cell-free protein production is the open-system environment which allows the direct quantification and/or analysis of protein functionality in an activity assay without purification. Alternatively, for some activity assays downstream processing via affinity purification may be needed (if an affinity tag is present). If you choose SDS-PAGE analysis, you can either take a small sample (1-3 µL) directly from your TXTL reaction, or – to reduce background signal – precipitate proteins with TCA/acetone or ammonium-acetate/methanol following a standard protocol.

It is very important to avoid condensation of water on the lid of the reaction tube as it considerably increases the concentration of myTXTL reaction components and can lead to poor or irreproducible performance. Incubation in an incubator and water bath/Lab Armor beads is best. Water facilitates a faster heat transfer than air and a water bath shows low temperature fluctuation, which should – combined with a closed environment with constant temperature surrounding the entire tube – lead to higher reproducibility and yield. myTXTL reactions also work well on an Eppendorf ThermoMixer with a lid.

We rcommend using the heavy and light chain templates at an equimolar concentration of 5 nM each whether they are plasmid or linear DNA templates.

Yes! Parameters that influence protein production efficiency are:

  • DNA design (promoter strength, position of affinity tag, TXTL elements)
  • DNA purity
  • DNA concentration
  • Incubation temperature, time, and vessel
  • Presence of folding helpers, chaperones, oxidizing agents

and should therefore be evaluated for optimization. Please also see our recommendations on these topics in the current myTXTL manual for your kit.