This manual introduces the use of Prime-It Fluor fluorescence labeling kit.
The Stratagene Prime-It Fluor fluorescence labeling kit generates directly fluoresceinated probes by incorporating fluorescein-linked dUTP into probe fragments. The process involves priming denatured DNA templates with random 9-mer oligonucleotides and extending them using exonuclease-free Klenow enzyme to incorporate a mix of nonmodified nucleotides and fluor-12-dUTP.
These fluoresceinated probes are designed for two primary applications: fluorescence in situ hybridization (FISH) experiments to localize genes on chromosomes, or the detection of nucleic acids immobilized on membranes using an antifluorescein antibody-based chemiluminescence system. This nonradioactive method offers increased safety and reduced background levels compared to traditional methods.
| Reactions supported per kit | 30 labeling reactions plus 5 control labeling reactions |
|---|---|
| Template DNA amount (recommended) | 25-50 ng per slide |
| Reaction buffer volume (sample) | 10 ul |
| Klenow enzyme amount (standard labeling) | 5 U |
The probes can be used for fluorescence in situ hybridization (FISH) experiments or to detect nucleic acids immobilized on membranes using an antifluorescein antibody-based chemiluminescence system.
All components should be stored at -20°C.