FAQs

We offer extraction services for DNA and RNA for multiple sample types. Some of the options for fresh samples are tissue (including plant leaves and seeds), cells, blood, and saliva. We also have the facilities and expertise for performing DNA extraction on degraded specimens, including bones, teeth, soft tissue, FFPE, and other sample types.

If you have special or sensitive samples, we may be able to offer extraction services for your project, depending on the circumstances. Please contact us directly to inquire about special service options.

Yes. We have a small cleanroom on-site where we perform all pre-PCR procedures. We can accommodate most common ancient, museum, or historical sample types, such as bones, teeth, and soft tissue. If you have a different or unusual sample type, please contact us to discuss your project needs in greater detail.

We offer NGS services on non-hazardous DNA or RNA extracts, whether from high-quality or degraded specimens. However, your samples should still meet our standard submission requirements for your type of service project, including relevant negative controls where applicable. Otherwise, your samples may be unable to pass our quality controls, or your project could be delayed. Please review the guideline documents in the Resources section.

Please note that if you are shipping your samples internationally which require any sort of import permit, you are responsible for identifying the permit and will incur a fee for any permit applications. Additionally, we require proof of ethics clearance for working with any human or human-derived samples.

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.

For fastest sample throughput, PCR products can be directly used as template material 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 our myTXTL Handbook and IDT App note for information about linear DNA template design requirements.

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 or bulk 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 all DNA plasmid and linear fragment sequences in our portfolio to allow users to optimize their designs with respect to 5′ and 3′ sequences that enable optimal expression for their application, ranging from single protein expression to complex gene circuits and metabolic pathways. Plasmid or linear DNA can be ordered online from various DNA synthesis companies.

Yes! Parameters that influence protein production efficiency are:

• Gene cassette construction (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 Template Design in the current myTXTL handbook.

Yes. The myTXTL master mix contains tRNAs for seven codons rarely used in E. coli to enable expression of eukaryotic proteins.

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.

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.

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.

Due to the small reaction volume of 12 μL, 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. This can lead to poor or irreproducible kit performance. If possible, incubation in an incubator and water bath/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. A PCR thermoblock did not perform well with tubes or a multiwell plate.

10 mM Tris/HCl pH 7.5.

Utilizing linear DNA templates greatly increases the speed of the design-build-test-learn cycle as laborious steps like cloning, transformation and purification are no longer necessary. This is particularly useful when working with a high number of variants of a single protein that need to be studied and validated. The reduced costs of linear DNA can also expand the sampled sequence space for protein designs compared to the plasmid format.

deGFP is a N- and C-terminally truncated version of the reporter eGFP that is more translatable in cell-free systems. The excitation and emission spectra as well as fluorescence properties of deGFP and eGFP are identical which enables the use of commercial eGFP protein to be used in a standard curve to quantify the deGFP in the reaction.

The myTXTL Linear DNA Expression Kit is based off our myTXTL Sigma 70 Master Mix Kit, but has been further engineered to efficiently produce soluble and membrane proteins using linear DNA templates without the need for nuclease inhibitors like GamS. Simply add linear DNA template to the optimized master mix to begin protein synthesis.

All P70 promoters originate from the lambda phage promoter for the repressor Cro with its two operator sites and are specific to the E. coli sigma factor 70. They differ in strength (P70a > P70d > P70b > P70c) due to mutations that were introduced at -35 and/or -10 regions.

We recommend setting the myTXTL GamS Protein concentration in a myTXTL reaction at 10 uM (myTXTL GamS stock solution is provided at 150 uM).

Steps of myTags Custom production using the Indexed Synthesis technique:

1. Probes are designed for each target region separately

2. All probe sequences (up to 27K total) are synthesized together

3. Using unique indexes, each individual probeset is separated into individual oligo pools via PCR deconvolution

4. Composition of oligo pools is verified with next-generation sequencing (NGS)

Note: Short index sequences remain present on the final probe oligos, but are short and not expected to interfere with downstream (F)ISH protocols.

We offer a range of available fluorescent and non-fluorescent label options; please contact us for current available options.

To better suit YOUR experimental needs, we now offer labeling as a service for our wide range of fluorescent and non-fluorescent label options, which can be applied to any of our upstream immortal pool synthesis products. This enables maximum flexibility to meet your dynamic research needs, such as the same probe oligos in multiple colors/tags or a new color/tag of a previously-ordered design.

Our new Labeling Service pairs perfectly with our new Indexed Synthesis options to create a fully custom, ready-to-use toolkit for your complex genome/transcriptome visualization projects.