Because high affinity nucleic acid binding dyes can affect DNA migration during electrophoresis, post-staining of gels with SYBR dyes is highly recommended. However, these dyes can be also be used as precast dyes (into the agarose gel). Please follow these recommendations:
Reduce the amount of DNA loaded to 1/8th of the recommended load, and use 0.5X SYBR dye in precast gels. SYBR dyes are 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 Purple 2-Log Ladder (NEB #N0550S) + 70 ul water + 20 ul 6X loading dye (NEB #B7024S) = 120 ul dilution at 25 ng/ul Load 5 ul at a time (125 ng).
It is preferable to wait a few minutes to add the SYBR 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.
SYBR is a registered trademark of Life Technologies Corporation
QUICK-LOAD is a registered trademark of New England Biolabs, Inc
TRIDYE is a trademark of New England Biolabs, Inc