Ideal labeling density will depend on the nucleic acid being labeled and the downstream application of the labeled nucleic acid. Using a high labeling density may be beneficial in direct nucleic acid tracking experiments, whereas a lower labeling density may be desirable in assays where the labeled nucleic acid must retain biological functionality. For example, transcription of highly labeled plasmid DNA may be inhibited compared to unlabeled plasmid DNA.1
With Label IT® nucleic acid labeling technology, the labeling density can easily be adjusted. In this Tip from the Bench, we discuss two methods for tweaking the labeling density.
Changes to the ratio of Label IT® Reagent to nucleic acid (v:w) affects the labeling density in a linear manner (Figure 1). For example, consider the starting recommendation of using 5 µl of Label IT® Reagent to label 5 µg of DNA (1:1). By altering the amount of Label IT® Reagent to 2.5 µl or 10 µl, while keeping the reaction incubation time and DNA amount constant, the labeling density will be halved or doubled, respectively. Note that using dramatically high Label IT® Reagent amounts (exceeding 4-fold than suggested) might lead to nicking of the nucleic acid and/or cause fluorescence quenching of Label IT® fluorophores.
Nucleic acid labeling density is proportional to amount of Label IT® Reagent and/or reaction incubation time used. Labeling density is easily controlled by adjusting the amount of Label IT® Reagent in the reaction or by adjusting the incubation time of the labeling reaction at 37°C.
At 37°C, the resultant labeling density is directly proportional to the Label IT® reaction incubation time between 15 minutes and 3 hours (Figure 1). In other words, by halving the suggested reaction time, the expected labeling density will be halved. Conversely, the labeling density can be doubled by doubling the reaction incubation time.