1. How do the Helixyte™ iFluor® dyes label nucleic acids?

The Helixyte™ iFluor® Labeling dyes features a reactive alkylating agent that strongly binds to nucleic acids through electrostatic interactions. This labeling process involves covalent modification (alkylation) of RNA or DNA, targeting the reactive heteroatoms found in any nucleotide within the nucleic acid polymer.

The Helixyte™ iFluor® Nucleic Acid Labeling Dyes reacts with the N7 of guanine residues to provide a stable coordination complex between the nucleic acid and the fluorophore label.

2. What types and sizes of nucleic acids are compatible with Helixyte™ iFluor® dyes for labeling?

You can label any purified nucleic acid with Helixyte™ iFluor® dyes, including plasmid DNA, siRNA, and oligonucleotides. We commonly label nucleic acids ranging from as short as 20 nucleotides to several kilobases in length. For optimal labeling efficiency, we recommend using oligonucleotides longer than 5 bases.

3. What labeling efficiency can I expect when using the Helixyte™ iFluor® Nucleic Acid Labeling Dye protocol?

Using the standard Helixyte™ iFluor® Nucleic Acid Labeling Dyes, we typically achieve one label every 20-60 base pairs, as estimated by a spectrophotometric assay for the fluorescent Helixyte™ iFluor® dyes. For information on calculating the labeling efficiency of your sample, see Question 10. Labeling densities are tailored to each specific application of the labeling dye.

4. Can I label dNTPs with Helixyte™ iFluor® dyes?

We do not recommend using Helixyte™ iFluor® dyes to modify dNTPs. Instead, we suggest using oligonucleotides longer than 5 bases to ensure efficient labeling.

5. How should I store Helixyte™ iFluor® dyes and labeled nucleic acids?

Tightly cap and store dried and reconstituted Helixyte™ iFluor® dyes and labeled nucleic acids at -20°C. Keep the Helixyte™ iFluor® dyes away from light and moisture to maintain their effectiveness. Improper storage can lead to reduced labeling efficiencies. Fluorescently labeled nucleic acids should also be stored shielded from light.

6. Can I scale the labeling reaction up or down?

Yes, you can adjust the labeling density, reaction volume, and the amount of nucleic acid labeled. To modify the labeling density, simply alter the ratio of the labeling reagent to nucleic acid or adjust the incubation time during the labeling reaction. You can also scale the volume of the labeling reaction up or down, using no more than 20% of the total reaction volume for the Helixyte™ iFluor® Nucleic Acid Labeling Dye, along with the labeling buffer of your choice diluted to a final concentration of 1X.

7. How would increased incubation times affect labeling efficiency?

When using the Helixyte™ iFluor® Nucleic Acid Labeling Dyes, we see a linear increase in labeling efficiency during the first 3 hours of incubation at 37°C. We recommend a standard reaction time of 1 hour at this temperature. However, because the labeling reaction is linear, you can achieve similar efficiency by using double the amount of Helixyte™ iFluor® Nucleic Acid Labeling Dye and incubating for just 30 minutes at 37°C.

8. How would increasing the amount of Helixyte™ iFluor® Nucleic Acid Labeling Dye (per 1 ug nucleic acid) affect labeling efficiency?

Generally, modest changes (2-4 fold) in the ratio of Helixyte™ iFluor® Nucleic Acid Labeling Dye to nucleic acid result in a linear effect on labeling efficiency. We do not recommend exceeding a 4-fold increase from the 1:1 (μl Helixyte™ iFluor® Nucleic Acid Labeling Dye : μg DNA) ratio, as this may increase the risk of nicking the nucleic acid template. When scaling the labeling reaction, the amount of Helixyte™ iFluor® Nucleic Acid Labeling Dye should not exceed 20% of the total reaction volume.

9. How can I prevent cross-labeling of additional DNA added to my sample after the initial labeling?

The covalent bonds between the label and the nucleic acid bases are highly stable, preventing cross-labeling or the transfer of labels to other species.

10. How can I estimate the number of fluorescent labels per nucleic acid molecule?

The simplest method to estimate the number of fluorescent labels on a DNA (or RNA) molecule is to measure the absorbance of the nucleic-dye conjugate at 260 nm (A260) and at the dye's λMAX (Adye). To quantify your labeled sample, you can calculate either the base to Helixyte™ iFluor® dye ratio or the pmol of dye per μg of nucleic acid.

11. Can I directly visualize nucleic acids labeled with Helixyte™ iFluor® Nucleic Acid Labeling dyes on a gel?

Yes, fluorescently labeled nucleic acids can be analyzed using agarose gel electrophoresis without ethidium bromide staining and subsequently visualized on a UV transilluminator. The bands may appear faint since the UV bulbs in the transilluminator emit light at around 300 nm, which is not the ideal excitation wavelength for the fluorescent labels.

12. What are the excitation and emission wavelengths for the Helixyte™ iFluor® Nucleic Acid Labeling dyes?

Following are the optimum excitation and emission wavelengths, as well as the correction factors at 260nm for the indicated Helixyte™ iFluor® fluorophores:

For more technical details, please refer to the respective product pages.

13. Does the quality and concentration of nucleic acid affect the labeling reaction?

Yes, it does. For optimal results, it is recommended to use purified nucleic acid in labeling reactions. For most applications, concentrated stocks of nucleic acid (approximately 1 mg/ml) are ideal.

14. Do dithiothreitol (DTT) or RNase inhibitors affect the labeling reaction?

DTT does not affect the labeling reaction. However, proteinaceous RNase inhibitors added before the labeling reaction may modestly compete with the nucleic acid. If the RNase inhibitors are introduced after the labeling is complete, they will not impact the product.

15. Can I transfect labeled DNA?

Yes, we have successfully delivered Helixyte™ iFluor® labeled DNA into various cell types using Transfectamine™ 5000 Transfection Reagent for DNA transport and gene expression studies. It’s important to note that highly labeled nucleic acids may inhibit transcription.

16. Can I transfect labeled siRNA?

Yes. We have successfully delivered Helixyte™ iFluor® labeled siRNA into a variety of cell types using our Transfectamine™ 7000 siRNA Transfection Reagent for siRNA tracking and gene suppression studies.

17. Can I use labeled DNA for DNA-binding studies such as gel-shift assays?

The Helixyte™ iFluor® Nucleic Acid Labeling Dye covalently modifies nucleic acid residues, which may interfere with the binding of certain proteins. You can adjust the ratio of Helixyte™ iFluor® Nucleic Acid Labeling Dye to DNA during the labeling reaction to achieve fewer labeled residues per DNA molecule. However, it is necessary to test the DNA in each specific binding application to assess its biological activity.

18. Can I use Helixyte™ iFluor® dyes for traditional hybridization applications?

Yes, you can. The Helixyte™ iFluor® dyes can be utilized to prepare non-radioactive probes for hybridization applications. Labeled DNA probes can be seamlessly integrated into your standard hybridization and detection protocols.

19. Can I use Helixyte™ iFluor® dyes for FISH applications?

Yes, Helixyte™ iFluor® technology is well-suited for preparing probes for FISH (fluorescence in situ hybridization) applications, which involve probing specific DNA sequences on chromosomes in metaphase spreads and/or interphase nuclei. Depending on the quantity of repetitive sequences in your chosen FISH probe, it may be necessary to add blocking DNA to minimize non-specific background signals. Common blocking DNAs include species-specific cot-1 DNA and sheared salmon sperm DNA.

20. Can I use Helixyte™ iFluor® dyes for microarray applications?

Yes, the Helixyte™ iFluor® Nucleic Acid Labeling Dyes can be optimized for labeling various microarray samples, including mRNA, cDNA, cRNA, and miRNA. Please note that additional detection reagents (not provided) may be necessary for microarray analysis. Other sample types, such as ribosomal RNA, bacterial RNA, and genomic DNA, can also be labeled using the Helixyte™ iFluor® Nucleic Acid Labeling Dyes.