The BrightStar Psoralen-Biotin Nonisotopic Labeling Kit provides an easy, efficient way to label nucleic acids used as probes for hybridization. Psoralens are planar tricyclic compounds that have a natural affinity for nucleic acids. Upon irradiation, the compounds intercalate between bases much like ethidium bromide. The kit can label oligonucleotides as small as 47-mers with concentrations as small as 5ng. In addition, this kit is extremely easy to use and comes with an informative and detailed manual.
The protocol suggests, for “best results”, begin with 0.5-50ng/ul of cleaned sample dissolved in TE buffer. The sample is denatured in a thin-walled PCR tube for 10 minutes at 100C and is then immediately quick-chilled in either liquid nitrogen or an ice water bath (remains frozen until the labeling process). The Psoralen-Biotin is initially resuspended in 33ul of the dimethylformamide in dim light and stored at 4C. Once the sample is thawed, 1ul Psoralen-Biotin is added to 10ul of sample (this can be increased proportionately). The sample is then transferred to a pre-chilled 96 well plate and it is irradiated with a 356nm UV light source for 45 minutes in a dark area. Ambion sells a relatively inexpensive hand held UV light that works great for this protocol. The sample is then cleaned by dilution to 100ul and placed in a microcentrifuge tube. The non-crosslinked Psoralen-Biotin is removed by butanol extraction (remove as much n-Butanol as possible, but traces of n-Butanol should not interfere with future reactions). An optional extraction with diethyl ether (not provided) can be done to remove trace amounts of the n-Butanol if desired. The biotinylated nucleic acids can be stored at –20C for short term or –80C for long term.
I have used this kit to label RNA for northern blot hybridization. Previously, I had used Dig-labeling that worked well, but was more time consuming. This labeling procedure gave me a well-labeled probe in about one hour. The kit allows for varied nucleic acid lengths and concentrations. The only limitation is the quality of RNA used as the starting material. The nucleic acids cannot be quantitated by absorbance after labeling since Psoralen absorbs at the same wavelength, so the control spot on the blot is the confirmation that the kit has worked. This kit made the labeling process much easier and faster than other labeling kits I have tried. The manual is very detailed and gives a brief protocol for experienced users as well as a detailed protocol for new users. It also comes with all the necessary reagents for labeling and cleaning. The long wave UV light needs to be supplied, but the Ambion portable UV light worked beautifully. I labeled one sample using this protocol but it could have just as easily and quickly labeled 96.
Overall, I think this kit is very useful when labeling nucleic acids and is a great replacement to the standard Dig-labeling or radiolabeling protocols.
Katrina Williams
Lab Specialist, Sr
McGuire VAMC
Richmond, VA