Unfortunately, not. However, studies have shown that supplementing cell-free systems with mixtures of reduced (GSH) and oxidized glutathione (GSSG), disulfide bond isomerase C (DsbC), protein disulfide isomerase (PDI) and/or chaperones (e.g. DnaK, DnaJ, GroEL, GroES) can promote the formation of disulfide bonds. In addition, pretreatment with iodoacetamide (IAM) to inactivate endogenous reductases which are present in the cell extract might also help (Review Article: Stech M & Kubick S, Antibodies 2015, 4, 12-33).
Yes, although it is not optimized or recommended for linear DNA templates. If using linear DNA with Sigma 70 MM, enhanced protein yields can be achieved by supplementing the master mix with our nuclease inhibitor GamS.
We always recommend keeping the thawing-and-freezing cycles as low as possible which might require aliquoting the original sample. However, myTXTL GamS Protein can be subjected to at least five thawing-and-freezing cycles.
PCR products can be directly used as DNA template for cell-free expression so long as the final glycerol concentration in the reaction is 0.1% or lower and the PCR reactions on average are making enough linear DNA to facilitate the protein yield needed. If a known fixed template concentration is preferred, PCR products should be subjected to a standard PCR clean up procedure with a final elution in molecular biology grade, nuclease-free water. This might also slightly boost the protein yield per reaction. Linear DNA can also be ordered and used directly in myTXTL from several commercial DNA suppliers. Please refer to the applicable myTXTL manual and our Application Note with IDT for information about linear DNA template design requirements, or reach out to techsupport@arbor.daicel.com with questions about specific sequence recommendations.
Unfortunately, this may lead to considerably decreased performance or even loss of function. To ensure highest kit performance, make sure to store the myTXTL kit master mix at -80 °C and freeze as soon as possible after usage.
Transformation efficiency depends on the quality of the competent cells. Make sure that cells were immediately frozen after preparation and stored at ≤ 80 °C. Please also note that for some cells, transformation efficiency drops drastically over time. Additionally, we advise to use E. coli strain KL740 for amplification of any plasmids containing σ70-specific promoter like P70a.
No. All our Toolbox 2.0 plasmids (except the positive control plasmid P70a-deGFP that comes with the myTXTL kit are meant for plasmid amplification in E. coli only. The degree of purity is NOT sufficient for efficient in vitro production. Please refer to the current myTXTL handbook for recommendations on preparation of plasmid templates for myTXTL reactions.
We share plasmid and linear DNA sequences that have been tested to work well in myTXTL, including kit plasmid controls, to allow users to optimize their designs with respect to 5′ and 3′ sequences that enable optimal expression for their application. Please contact techsupport@arbor.daicel.com with any questions.
Yes. The myTXTL master mix contains tRNAs for seven codons rarely used in E. coli to enable expression of eukaryotic proteins.
Yes! That only requires the addition of the plasmid coding for T7 RNA polymerase under transcriptional control of a σ70-specific promoter, e.g. P70a-T7rnap and possibly your inducer like IPTG if it is an inducible promoter. The optimum concentration of P70a-T7rnap is usually between 0.1 nM and 1 nM. Higher concentration normally does not increase protein yield. The more important parameter for efficient protein expression is the concentration of the plasmid that encodes for your protein of interest downstream of the T7 promoter, which will be most likely in the range of 5-20 nM.
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