10 mM Tris/HCl pH 7.5.
Using 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).
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.
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