Nucleic Acid Labeling Support—Troubleshooting

Here are some possibilities and our suggestions for addressing them:

• Check the age of the Alexa Fluor amine-reactive dye and how it has been stored. The dyes are sensitive to hydrolysis and can lose reactivity if exposed to moisture or water. They should be stored as a powder and dessicated to protect the dye from water. Anhydrous DMSO should be used for dissolving the dye, and once the dye is dissolved in DMSO, it should be used right away, since it will be more sensitive to hydrolysis and less stable in solution. The reactive dye should also be protected from light during storage. When stored dessicated and protected from light in powder form, dyes are stable for at least 6 months; however, dyes older than this may have hydrolyzed and no longer be reactive. 
• For some reactions, a larger dye-to-oligonucleotide molar ratio may be necessary, so you may need to use more dye or less oligonucleotide in the reaction. 
• The reaction works best at a slightly basic pH so that the amine on the oligonucleotide is deprotonated, so a 0.1 M sodium borate, pH 8.5 labeling buffer should be used for the reaction. For best results, other buffers should not be used, since they may not have the correct pH or may contain components that interfere with the reaction. 
• Make sure there are no proteins or primary amines in the reaction. The amine-modified oligonucleotide should be extracted and purified before the reaction to remove any amines such as Tris, triethylamine, ammonium salts, glycine, BSA, or other amine-containing molecules since these will react with the amine-reactive dye and reduce the efficiency of the reaction. 
• 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 dye 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.

• 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.