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.
Atto-550 is our most popular dye labeling option, followed by Atto-488, Atto-594, and Atto-647N. Biotin, Digoxigenin and 6FAM are also popular options that work well in multi-color FISH assays.
We may be able to accommodate other labeling options, please contact us for availability.
The number of assays per library depends on a number of factors including the probe density of your library, the size of your target region, the number of probes in the library, and the FISH protocol. Generally we recommend starting with 10pmol of labeled probes per standard FISH slide and then modifying the input amount based on the initial results.
The latest recommended protocols for both labeled and immortal probe libraries can be found in the Resources tab, but in general, myTags FISH libraries are compatible with most FISH protocols. Please contact us for specific recommendations.
We can often accommodate customer-designed probes into the myTags labeling framework. Please contact us for recommendations on the design parameters and other information before designing your probe sequences.
Yes, we can synthesize immortal probe libraries that can be labeled using the Oligopaints labeling method. Please note these probe libraries are not compatible with the standard myTags labeling protocol due to sequence requirements of the Oligopaints method.
We generally ask for up to 3-4 weeks after an order is placed to ship myTags libraries.
We recommend using the myTags labeling protocol with myTags immortal libraries for perfroming the labeling process in your own lab. If you would like to use a different protocol, one of our scientists would be happy to provide assistance to ensure success.
Probes are available in 2 configurations to detect both the positive (+) strand and negative (-) strand sequences:
Positive (+) strand probes– SARS-CoV-2 is a class IV RNA virus with an ssRNA genome (positive strand). The probes to detect the (+) strand will hybridize to full-length genomic (+) strand RNA as well as subgenomic (+) strand RNAs made during replication.
Negative (-) strand probes– Detection of the (-) strand is a hallmark of active viral replication. The probes to detect the (-) strand will hybridize to the full-length (-) strand as well as subgenomic (-) strand RNAs. In experimental applications where viral infection is not controlled, additional confirmation of SARS-CoV-2 presence via PCR is recommended.
NOTE: The positive (+) and negative (-) strand probes are not designed to be used in the same hybridization experiment. If co-hybridization is required for your experimental design, please contact us for a customized probe solution.