Nucleic Acid Labeling Support—Troubleshooting

Insufficient removal of free dye could lead to high background. Try purifying the oligonucleotides by HPLC or gel electrophoresis to ensure removal of unreacted dye.

• 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.

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.

Nucleic acids cannot be quantitated by absorbance at 260 nm after using a BrightStar® Psoralen-Biotin Nonisotopic Labeling Kit because psoralen absorbs at this wavelength, interfering with an accurate reading and potentially destroying the crosslinks that connect biotin to the nucleic acid. Quantitation is recommended before labeling with the BrightStar® Psoralen-Biotin Nonisotopic Labeling Kit. Yield from the procedure will be 90–100%, with a slight loss resulting from the butanol extraction.

If the positive controls provided with the kit are detectable at 100 fg to 1 pg, indicating that the BrightStar® Psoralen-Biotin Nonisotopic Labeling Kit and the biotin detection system are both performing as expected, but the test nucleic acid appears not to have been labeled efficiently, we recommend running the material on a gel to see if it is intact. If the test nucleic acid is intact as determined by gel electrophoresis, then there may be inhibitors present that are interfering with the labeling reaction. The presence of free nucleotides from an in vitro transcription reaction or from PCR could cause this problem. Free nucleotides can be removed by precipitation with salt/ethanol or isopropanol, followed by a 70% ethanol wash. There are a number of factors that are important for the efficient labeling of nucleic acids using the BrightStar® Psoralen-Biotin Nonisotopic Labeling Kit. IT=he labeling procedure must be followed exactly as specified. Some of the crucial variables are listed below.

1. Make sure that the correct wavelength of UV light is used. Only long-wave UV (365 nm) works. Short-wave (254 nm) and medium-wave (310 nm) UV should not be used; short-wave radiation is particularly damaging to nucleic acids. 
2. The reaction must be done under reduced lighting. If desired, the lights can be turned off completely after the reaction has been set up. 
3. Proper heat denaturation is critical for efficient labeling of DNA (see steps 2–3 on page 7 of the manual). Make sure the heat denaturation is done at 100°C for at least 10 minutes. Quick-chill the sample immediately in a dry ice/alcohol bath. (Liquid nitrogen or an ice water bath could be used instead.) 
4. The pH of the nucleic acid solution must be between 2.5 and 10. 
5. The salt concentration must be below 20 mM. 
6. We have found that irradiation for as little as 15 minutes is often sufficient to produce probes with high specific activity. We do not recommend irradiating the sample for more than an hour.

The following suggestions may help reduce the background when using a probe labeled using the BrightStar® Psoralen-Biotin Nonisotopic Labeling Kit in blot hybridizations:

1. Filter the probe and hybridization solution before use. 
2. Use no more than the probe concentrations recommended in Section III.D. Using BrightStar® Psoralen-Biotin Labeled Probes on page 9 of the manual. 
3. Increase the SDS concentration in the hybridization and wash buffers (up to a maximum of 2%). 
4. Increase the blocking time prior to and after incubation with the antibody: conjugate solution. 
5. Use a lower concentration of antibody: conjugate solution. 
6. Increase the wash time and/or the number of washes after the conjugate binding and blocking steps.

There may be an inhibitor in the nucleic acid substrate. If needed, clean up the nucleic acid you want to label with a phenol: chloroform: isoamyl alcohol extraction followed by spin-column purification. Alternatively, the 5’ end of the RNA may be inaccessible because of tertiary structure. In this case, try heating the RNA to 90°C for 5 minutes, then place it immediately on ice just before the reaction.

We recommend cleaning up the kinase reaction using a phenol/chloroform extraction followed by ethanol precipitation, then eluting the DNA. If this doesn’t help, further purify the reaction by spin-column chromatography or denaturing polyacrylamide electrophoresis.