According to the manufacturer, because high affinity nucleic acid binding dyes can affect DNA migration during electrophoresis, post-staining of gels with GelRed is highly recommended. Post-staining with GelRed results in superior sensitivity and eliminates the possibility of dye interference with DNA migration. While agarose gels can be precast with GelRed, GelRed may affect the migration or resolution of some DNA ladders in precast gels. To optimize the results when using GelRed as a precast dye, follow these recommendations:
Reduce the amount of DNA Ladder loaded. GelRed is much more sensitive than EtBr. Blown out or smeared bands can be caused by overloading, which is frequently observed with DNA ladders.
- For our standard DNA ladders (not ready-to-load), reduce the amount of DNA loaded to 1/8th of the recommended load, and use 0.5X GelRed in precast gels. Loads between 60 and 125 ng of DNA ladders yield the best results (as opposed to 500 to 1,000 ng as recommended for regular gels with ethidium bromide).
- For our ready-to-load DNA ladders, such as Quick-Load® or TriDye™ DNA Ladders, dilute the DNA ladders by 1/4th, using 1X loading buffer. Avoid diluting the ladders in water only, as they may become difficult to load, and float out of the wells.
Example 1: 30 µl Quick-Load 1 kb Ladder (NEB #N0468S) + 70 µl water + 20 µl 6X loading dye (NEB #B7021S) = 120 µl dilution at 12.5 ng/ul. Load 5 µl at a time (62.5 ng).
Example 2: 30 µl Quick-Load 2-Log Ladder (NEB #N0469S) + 70 µl water + 20 µl 6X loading dye (NEB #B7021S) = 120 µl dilution at 25 ng/ul. Load 5 µl at a time (125 ng).
Use our Fast DNA Ladder (NEB #N3238S), which does not require any dilution, and is ready-to-load for use with GelRed precast gels. Load 5 µl per lane (125 ng). Size range: 50 bp to 10 kb.
Cool the agarose before adding the GelRed dye. Although the manufacturer claims that GelRed can be added while the gel solution is still hot, in our experience, it is preferable to wait a few minutes to add the GelRed dye to the molten agarose. Adding it to a cooled agarose solution (above gelling temperature, 45°C) gives better results.
Always run a freshly made agarose gel, for a single run only. Re-running the gel will yield poor results.
Ensure the GelRed has been stored in the appropriate conditions (at room temperature and protected from light). Make sure the GelRed is not past its expiration date, as it is only stable for one year.