How does sybr safe work

SYBR Safe is a cyanine dye used as a nucleic acid stain in molecular biology. SYBR safe stain is specifically formulated to be a less hazardous alternative to ethidium bromide that can utilize either blue light or uv excitation. SYBR Safe. It is marketed as less harmful than ethidium bromide, but this is debatable. Its major advantage is that it is as sensitive as ethidium bromide but does not require UV light for visualization.

SYBR Safe stain is specifically formulated to be a less hazardous alternative to ethidium bromide that can be used with either blue-light or UV excitation. How much DNA should be loaded per well of an agarose gel? The amount of DNA to load per well is variable. The least amount of DNA that can be detected with ethidum bromide is 10 ng.

DNA amounts of up to ng per well will result in a sharp, clean band on an ethidium bromide stained gel. When bound to DNA, the dye chances structurally and becomes less mobile, causing its energy to be released as fluorescence. The fluorescence increases along with the concentration of DNA.

The minimum amount visible using EtBr is 10ng per band or even less though I never saw anything below 5ng per band. The concentration of the gel is mainly important for the DNA separation - the bigger your DNA is, the lower is the percentage of the gel.

GelRed is an intercalating nucleic acid stain used in molecular genetics for agarose gel DNA electrophoresis. When exposed to ultraviolet light, it will fluoresce with an orange color that strongly intensifies after binding to DNA. This unique product stains DNA deep blue in both agarose and polyacrylamide gels, providing vivid, consistent results. At Biotium, innovation is at the very heart of what we do every day.

Use an alternative DNA stain! Ethidium bromide is not your only option. In this article, we will compare the available DNA stains that can be used in electrophoresis to clarify the options available to you.

The classic DNA stain. Ethidium bromide EtBr is a flat molecule that fits between adjacent base pairs intercalates in the DNA double helix. It has UV absorbance maxima at nm and nm and can also absorb energy from nucleotides excited at nm. The fluorescence of EtBr is significantly higher when intercalated than it is in aqueous solution.

Protocol: Can be used in the gel at or as a post-stain at a concentration of 0. However, it has the advantage that is detectable in the visible range. Destaining in water may be required for maximum sensitivity. Protocol: Post strain only, in 0. Detection: Visible light. Sensitivity: 40— ng bands are reported to be detectable after de-staining. Toxicity: Non-mutagenic.

Somewhat toxic if ingested. References: Yung-Sharp, D. Crystal violet intercalates into DNA in a similar manner to ethidium bromide but is less mutagenic. There is a vast improvement in cloning efficiency of DNA fragments stained with SYBR Safe stain and visualized using blue-light versus the same fragments stained with ethidium bromide and exposed to UV light. Place the gel in a plastic container, such as a pipet-tip box lid or a household food-storage container.

Do not use a glass container, because the dye in the staining solution may adsorb to the walls of the container, resulting in poor gel staining. A 50 mL volume is sufficient for staining most standard minigels. To stain larger gels, increase the volume of staining solution in proportion to the increased gel volume, and ensure that the gel is fully immersed during staining.

Incubate for 30 minutes. Cover the gel and the staining solution with aluminum foil or place them in the dark to protect from light. Continuously agitate the gel on an orbital shaker at 50 rpm. No destaining is required. As with precasting gels with ethidium bromide, the mobility of nucleic acid fragments in the gel may be somewhat slower when run in these gels compared to their mobility in the gel without stain.

Run the gel. No post-staining or destaining is needed. You can view stained gels using a standard nm transilluminator, a nm epi- or transilluminator, or a blue-light transilluminator such as the Safe Imager 2.

Note: If bands from the SYBR Safe stained gel are to be excised and used in a ligation reaction, we recommend that the gel is illuminated using blue-light source i. Using this film and one of these filters, SYBR Safe DNA gel stain provides the same detection sensitivity as ethidium bromide and an appropriate filter.

This is especially valuable when performing exposure-intensive protocols like cutting bands out of gels. SYBR Safe stain offers excellent sensitivity in nucleic acid visualization with either UV excitation or blue-light excitation.