Can SYBR Green dye replace EB dye for DNA gel? How effective and safe are they

I suggest post staining for PCR product identification. Although pre dyeing is convenient, the dye will bind to DNA and may slightly alter the migration rate, especially for small fragments or thicker gels; The background of post dyed glue is usually more uniform and the stripes are clearer. 1.5% glue is suitable for post dyeing: After running the glue, put it into freshly prepared 1 × TAE or TBE, add dye to the recommended concentration, shake in the dark for 20-40 minutes, and if necessary, decolorize for 10-20 minutes. If the strip is weak, try different exposure times or increase the sample size to 5ul, but do not exceed the capacity of the gel hole.

The dilution ratio must follow the manufacturer's instructions. The concentration of SYBR Green I nucleic acid dye can vary significantly between different manufacturers, so the 1:10,000 experience with EB cannot be directly applied. The post-staining concentration we commonly use is approximately 1×. After dilution, stain for 30 minutes protected from light. If the background is high, shorten the staining time or extend the destaining time. Prepare a small amount of staining solution first to avoid waste. Either 1× TAE or TBE can be used as the aqueous buffer, but high-salt buffers may affect dye binding, so consistency must be maintained. Excessive staining time will result in high background, while insufficient staining time will cause weak bands to be blurry. It is recommended to run a DNA ladder of known concentration on the same gel as a reference.

SYBR Green I can replace EB for DNA gel staining, but imaging conditions need to be re-optimized. It can be excited under UV, but many labs use blue-light gel cutters, which provide better signal and lower DNA damage and risk. Typically, 5 µL of PCR product run on a 1.5% agarose gel is visible, but band brightness depends on PCR yield and dye lot. When photographing under UV, exposure time usually needs to be re-determined; SYBR Green I sensitivity is similar to or slightly higher than EB, but background may be more pronounced. Run a preliminary test gel first to confirm voltage and time to avoid running the bands too far.

When cutting and recycling glue, if SYBR Green I binds to DNA and the UV irradiation time is too long, it may cause DNA damage or dye residue that affects subsequent enzyme reactions. It is recommended to cut the adhesive under blue light. If only ultraviolet light is available, try to cut as quickly as possible, use a long wavelength transilluminator, and reduce the irradiation. Most commercial adhesive recycling kits can remove most of the dyes, but for safety reasons, the concentration and 260/280 should be tested after recycling, followed by a trial amplification. In terms of safety, SYBR Green I is less irritating than EB, but gloves should still be worn to avoid skin contact and inhalation of the concentrated solution; Collect waste liquid according to laboratory chemical waste liquid.