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View additional product information for ChromaTide™ Texas Red™-12-dUTP - FAQs (C7631)
10 product FAQs found
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.
Although we have a protocol for incorporation using PCR, we do not recommend PCR as the best method for incorporation of any of our fluorescent or modified nucleotides. With PCR, incorporation rates are very low. We had some success with Taq polymerases in the past; however, many of the modern DNA polymerases are higher fidelity; as a result, they do not incorporate modified nucleotides very well. This paper below provides a comparison of commercially available polymerases and PCR incorporation efficiency related to type of dye or modification:Anderson JP, Angerer B, Loeb LA (2005) Incorporation of reporter-labeled nucleotides by DNA polymerases. Biotechniques 38:257–264.
ChromaTide dUTPs are most efficiently incorporated into DNA using nick translation, random primer labeling, end-labeling with terminal deoxynucleotidyl transferase (TdT), and reverse transcription. ChromaTide UTPs can be incorporated into RNA using in vitro transcription. You can find example protocols using these enzymatic reactions below. Specific fluorescent ChromaTide nucleotides may not be appropriate for certain enzymatic methods, so we recommend consulting Table 1 in the ChromaTide product manual prior to purchase. Some ChromaTide products have been discontinued, so not all of the listed ChromaTide nucleotides may be currently available (http://tools.thermofisher.com/content/sfs/manuals/mp07603.pdf, http://tools.thermofisher.com/content/sfs/manuals/td07604.pdf, http://tools.thermofisher.com/content/sfs/manuals/td07605.pdf).
The ChromaTide dUTP and UTP nucleotides are modified at the C-5 position of uridine via a unique alkynylamino linker, which provides a spacer between the nucleotide and the dye to reduce interactions between them. Several of these nucleotides also contain an additional spacer, further separating the label from the nucleic acid. The number in the product name, e.g., the “12” in fluorescein-12-dUTP, indicates the net length of the spacer, in atoms.
ChromaTide dye-labeled dUTP and UTP nucleotides can be used to synthesize labeled nucleic acid probes without the need for hazardous and expensive radioisotope-labeled nucleotides. You can incorporate these nucleotides using standard molecular biology techniques, and then use the labeled probes for in situ hybridization, microarrays, or blotting. ChromaTide dye-labeled nucleotides are available with different fluorescent colors to facilitate multicolor analysis.
You will need to confirm with the manufacturer of the restriction enzyme you are using to find out whether or not it can recognize dUTP-containing DNA.
Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.