The myTXTL Pro Kit is intended for expression of proteins that do not require disulfide bonds. It also has the highest yield of our two kits. The Antibody/DS Kit is intended for the expression of proteins that do contain disulfide bonds such as antibodies and enzymes. The yield of the Antibody/DS kit varies with the protein expressed, but for deGFP it is about 30-50% the yield of the Pro Kit. This is still enough protein for most downstream applications.

For work involving proteins that require disulfide bond formation we recommend our myTXTL Antibody/DS Kit. It is possible that some proteins with just 1 disulfide bond, such as VHH/Nanobodies, may express and show activity in the myTXTL Pro Kit, but generally speaking disulfide bond containing proteins will have the highest yields and best activity in the myTXTL Antibody/DS Kit.

If you were using any of our 3 original myTXTL kits, those will be discontinued in August 2024. The myTXTL Pro Kit is now the kit that will serve your needs as it has all the properties of the old kits combined into one: it supports linear and plasmid DNA as well as all E. coli promoters and also enables T7 promoter-based expression through the addition of the Pro Helper Plasmid that expresses the T7 RNA polymerase. All Pro Kits include the Pro Helper Plasmid and a T7 deGFP Positive Control Plasmid.

If you are interested in expressing proteins that contain disulfide bonds, the myTXTL Antibody/DS Kit is what you need.

Yes, myTXTL reactions have been conducted from 2 to 100 uL volume, but above 50 uL we recommend shaking and/or switching to a reaction vessel with higher surface:volume ratio to allow proper oxygenation of the reaction mix. myTXTL reactions are very sensitive to the amount of dissolved oxygen. If using over 50 uL volume, a flat-bottomed ELISA, deep well plate or tissue culture plate may be advised along with shaking at 650 RPM. We advise testing such a setup with one of the positive control plasmids, such as T7 deGFP. The key is to balance oxygenation and avoid the reactions drying out due to too much surface area. Please refer to the appropriate kit manual for additional guidance on scaling up reaction volume.

As the myTXTL platform relies on the endogenous transcription and translation machinery of E. coli, a functional gene cassette must contain a promoter that can be transcribed by E. coli RNA polymerase and associated transcription factors (primarily Sigma 70) or by a T7/T3 RNA polymerase if those polymerases are expressed from a helper plasmid (available in our Toolkit). The ribosomal binding site should also be compatible with E. coli translation machinery. For more general advice on how to construct a functional gene cassette, please refer to the current myTXTL handbook.

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. All kits come with a Helper plasmid to express the T7 RNA polymerase and enable transcription from T7 promoters. If a promoter is used that is recognized by the E. coli RNA polymerase, then this helper plasmid is not needed. If using a plasmid with an inducible promoter, such as the pET vectors, you need to add your inducer IPTG at 1 mM to the reaction and you may want to explore plasmid concentration in the range of y your inducer like IPTG if it is an inducible promoter.

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.