- Reduce the amount of DNA loaded to 1/8th of the recommended load, and use 0.5X GelRed in precast gels. GelRed is much more sensitive than EtBr. Blown out or smeared bands can be caused by overloading, which is frequently observed with DNA ladders.
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 ul Quick-Load 1 kb Ladder (NEB #N0468S) + 70 ul water + 20 ul 6X loading dye (NEB #B7021S) = 120 ul dilution at 12.5 ng/ul Load 5 ul at a time (62.5 ng).
Example 2: 30 ul Quick-Load 2-Log Ladder (NEB #N0469S) + 70 ul water + 20 ul 6X loading dye (NEB #B7021S) = 120 ul dilution at 25 ng/ul Load 5 ul 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 ul per lane (125 ng). Size range: 50 bp to 10 kb.
- 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.
- Make sure 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 stable for one year.