Because high affinity nucleic acid binding dyes can affect DNA migration during electrophoresis, post-staining of gels with SYBR dyes, such as SYBR Safe, is highly recommended. However, these dyes can be also be used as precast dyes (into the agarose gel). To optimize the results when using SYBR dyes as precast dyes, please follow these recommendations:
- Reduce the amount of DNA loaded to 1/2 of the recommended load. Blown out or smeared bands can be caused by overloading, which is frequently observed with DNA ladders.
Loads between 250 and 500 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 1/2, 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: 60 ul Quick-Load Purple 2-Log Ladder (NEB #N0550S) + 50 ul water + 10 ul 6X Purple Loading Dye No SDS (NEB #B7025S) = 120 ul dilution at 50 ng/ul
Load 10ul at a time (500 ng).
- Please follow the manufacturer’s recommendations and protocols carefully.
- Avoid using loading dyes containing SDS, as SDS can cause interference when using SYBR dyes into the gel. We recommend using our Gel Loading Dye, Purple (6X), no SDS (NEB #B7025S) for this application.
- We recommend using TBE buffer instead of TAE buffer as the TAE buffer can accelerate the migration of the SYBR dye in the gel and result in the very bottom of the gel having a lower concentration of stain. This effect can be partially counteracted by adding SYBR® Safe DNA gel stain to the running buffer. Also, the SDS interference can be increased when using TAE buffer in the gel.
- Always run a freshly made agarose gel, for a single run only. Re-running the gel will yield poor results.