ChromaTide™ Alexa Fluor™ 488-5-dUTP

You can try to purify the ChromaTide labeled probe with an appropriate spin column-based method to remove unincorporated ChromaTide nucleotides. Ethanol precipitation may not efficiently remove the unincorporated ChromaTide nucleotides, so a spin column will need to be used.

- Check the base-to-dye ratio to determine the level of incorporation of the ChromaTide nucleotides. Since fluorescent detection may be affected by underlabeling, overlabeling, instrument sensitivity, or other factors, the base-to-dye ratio is a better indicator of incorporation efficiency.
- ChromaTide nucleotides may not have been incorporated well in the enzymatic labeling reaction. Make sure that the enzymatic method used is compatible with the particular fluorescent ChromaTide nucleotide, since some methods may not be appropriate for all applications. You may also need to further optimize the enzymatic incorporation method, for example by optimizing enzyme concentration, incubation time, concentration, and ratio of labeled and unlabeled nucleotides. For PCR, a lower fidelity polymerase may give higher incorporation rates; however, incorporation rates will be generally low using PCR.
- Check the fluorescent filter used for detection to make sure it is compatible with the dye. You can also test a small drop of the undiluted fluorescent ChromaTide nucleotide in your filter to make sure you can image the dye alone before it is conjugated to the oligonucleotide. The fluorescence emission of Alexa Fluor 647 is not visible by eye and will require a far-red imaging system for detection.

No, they are not cell permeant so they are only suitable for in vitro incorporation methods. The fluorescent dyes and phosphate groups are too highly charged to allow the nucleotides to penetrate the membrane of an intact cell. Nonfluorescent nucleosides without phosphates such as EdU, EU, or BrdU can be used for live cell nucleic acid incorporation studies.

The base-to-dye ratio is determined by measuring the absorbance of the nucleic acid at 260 nm and the absorbance of the dye at its absorbance maximum. Using the extinction coefficients for the appropriate dye and nucleic acid, you can then calculate the base-to-dye ratio for the labeled nucleic acid using the Beer-Lambert law. Detailed instructions can be found in these product manuals: (http://tools.thermofisher.com/content/sfs/manuals/td07604.pdf, http://tools.thermofisher.com/content/sfs/manuals/td07605.pdf).

The average incorporation is one dye for every 100-150 bases, so the ChromaTide fluorescently labeled nucleotides typically produce the lowest labeling rates of the nucleic acid labeling methods we offer.