ARES™ Alexa Fluor™ 647 DNA Labeling Kit

Different preparations of RNA will certainly give different results. Most of the time, the mRNA is significantly degraded. The enzymatic incorporation of aminoallyl-dUTP (AA-dUTP) should not differ from reaction to reaction. If there are differences, it has to be due to the RNA or the method. AA-dUTP incorporation is no different than that of a dye-nucleotide conjugate, and should be more efficient and uniform. Here are a couple of suggestions:

1) cDNA may have been lost prior to labeling. Add 1 µL of glycogen (molecular biology grade), containing 10-20 µg, to the cDNA before precipitating it with ethanol.

2) Make sure to add sodium acetate as the salt and not ammonium acetate. After pelleting the cDNA, resuspend it in 5 µL water.

3) If generating long cDNAs, it will help to heat-denature the sample. Heat it at 95°C for 5 minutes and then put it on ice for a few minutes. Then centrifuge it for a few minutes just prior to the labeling reaction.

4) You want the tube to be at room temperature for the labeling reaction. Add the 3 µL of buffer and mix it in. Then add the dye and vortex it vigorously for at least 15 seconds.

Unfortunately, Alexa Fluor 633 does not label nucleic acids well because of the dye's chemical structure. Furthermore, DNA probes labeled with Alexa Fluor 633 do not form stable hybrids in nucleic acid hybridization assays.

An ARES-labeled oligonucleotide should survive at 95°C for 5 minutes.

Long-term storage for the ARES-labeled probes can be done in just about any kind of buffer, TE, formamide, hybridization buffer, or ethanol. We suggest using your normal storage conditions as long as you protect the probes from light. ARES conjugates are very stable.

At the same dye-to-base ratio, Alexa Fluor dyes exhibit higher intensity and reduced self-quenching at higher labeling ratios.