Tips from the Bench: Nucleic Acid Labeling Tip

Adjust Labeling Density Using Label IT® Reagents

Specific research applications may require different nucleic acid label densities for optimal performance. While high labeling densities often yield optimal results in direct nucleic acid tracking experiments, lower labeling densities might be desirable in assays requiring biological functionality. For example, transcription of highly labeled plasmids may be inhibited compared to unlabeled plasmids (1). With Label IT® technology, the amount of reagent and the reaction time can be easily adjusted enabling optimization of labeling density for user-defined applications.

Vary Label IT® Reagent Amount

With the general Label IT® Nucleic Acid Labeling Kits, modest changes (2–4 fold) in the ratio of Label IT® Reagent to nucleic acid affect the labeling efficiency in a linear manner (Figure 1). The starting recommendation is to use 5 µl of Label IT® Reagent to label 5 µg of DNA. By altering the amount of Label IT® Reagent to 2.5 µl or 10 µl while keeping the reaction incubation time constant, the labeling density will be halved or doubled, respectively. Dramatic changes in Label IT® reagent amounts (exceeding 4-fold) might lead to nicking of the nucleic acid and/or cause fluorescence quenching in case of Label IT® fluorophores.

Alter Reaction Incubation Time

In general, labeling density is directly proportional to incubation time (at 37ºC) between 15 minutes and 3 hours (Figure 1). For example, the general Label IT® Nucleic Acid Labeling Kits recommend incubating the labeling reactions for 1 hour at 37ºC. By decreasing the reaction time to 30 minutes, the labeling density can be halved. Conversely, the labeling density can be doubled by extending the reaction time to 2 hours.

Note: Labeling densities may be different if you are using an application-specific labeling kit that has been optimized to achieve the optimal labeling density for the specific application with a 1 hour labeling reaction at 37°C.

Flexible Labeling Reaction Conditions: Titration of Labeling Density

Flexible Labeling Reaction Conditions. 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 37ºC.

Reference

  1. Efficient in vitro and in vivo expression of covalently modified plasmid DNA. Slattum PS, Loomis AG, Machnik KJ, Watt MA, Duzeski JL, Budker VG, Wolff JA, Hagstrom JE. Mol Ther. 2003 Aug;8(2):255-63.

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