Fluorescent labels (Cy®3, Cy®5, TM-Rhodamine, CX-Rhodamine, Fluorescein)
The relative density of fluorescent labels on purified, labeled nucleic acid can be assessed by:
- Gel electrophoresis without ethidium bromide - Larger molecules such as plasmids and long PCR products should be analyzed using 1% agarose gels whereas small nucleic acid samples such as siRNA or oligonucleotides are best analyzed using acrylamide gels. The nucleic acid will appear faint under UV illumination because the transilluminator emits at approximately 300 nm, which is not optimal for the fluorescent labels (see Table 1). With subsequent ethidium bromide staining, a gel shift may be detected (relative to unlabeled nucleic acid), indicating successful labeling (see Figure 1).
- Spectrophotometric absorbance at lambda max - Several micrograms of labeled nucleic acid may be required to generate significant lambda max absorbance readings (see Table 1).
- Fluorimetric detection at the specific excitation and emission wavelengths - Refer to Table 1.
- Fluorescent microscopy - Spot dilutions of labeled nucleic acid onto a glass slide and view with fluorescent microscope.
- In Vitro tracking - Transfect labeled DNA into a common cell line, such as COS-7, HeLa, or NIH 3T3 to ascertain detection efficiency and optimal parameters.
Epitopes (Biotin, DNP, Fluorescein)
For a qualitative assessment of epitope labeling, the following methods can be used:
- Gel shift - With ethidium bromide staining, a gel shift may be detected (relative to unlabeled DNA), indicating labeling of the nucleic acid (see Figure 1).
- Dot blots - Labeled nucleic acid can be spotted on a nylon membrane and detected with epitope-specific antibody or streptavidin conjugates.