Ethidium Bromide (EtBr) has become the most used dye for nucleic acid staining due to its low cost and high sensitivity. Despite of its effectivity as a dye, EtBr is determined as highly mutagenic reagent. It requires safe conditions for work in the laboratory to eliminate the hazardous effect and special procedures for decontamination and waste disposal. All these precautions lead to an increase of the costs for nucleic acid analyses.
- 1.Is there a non-hazardous dye for nucleic acid staining available on the market?
Yes. The American company Biotium developed GelRed and GelGreen fluorescent agarose gel stains. According to the results of independent laboratories, these dyes are the most sensitive and completely safe DNA gel stains on the market.
- 2.Why should I use GelRed instead of EtBr?
GelRed nucleic acid stain has many advantages in comparison with EtBr:
- It is the most sensitive fluorescent red DNA gel stain;
- Classified as non-hazardous waste in compliance with CCR Title 22 Hazardous Waste Characterization;
- It can replace the EtBr in staining procedures for dsDNA, ssDNA or RNA in agarose gels or polyacrylamide gels;
- Can be used for post staining or precast in agaroses;
- Visualized with UV transilluminators using EtBr detection settings
- Three independent testing services proved that GelRed cannot get through latex gloves or cell membranes;
- It is categorized as non-cytotoxic and non-mutagenic dye at concentrations above the working values used in gel staining;
- It is available for purchase as a 10,000X stock, ready-to-use 3X solution and in 6X loading buffer format.
- 3.What are the steps for post staining and precast staining procedures?
For Post-staining with GelRed, you need to follow the steps below:
- §Run gels as it is described in your standard protocol;
- §GelRed should be diluted in electrophoresis buffer to 3X concentration.
- §Carefully place the gel in a suitable container such as a polypropylene staining tray. The GelRed 3X staining solution should be added in enough volume to submerge the gel.
- § Agitate the gel gently at room temperature for half an hour approximately.
- Note: De-staining is not required. However, the gel can be washed in water to reduce background if necessary.
- §Make an image of the gels using an EtBr filter
- The precast staining protocol for agarose includes the following steps:
- §The GelRed should be diluted in concentrated electrophoresis buffer to 1X concentration.
- §Add agarose powder, heat and mix thoroughly to obtain a homogenous GelRed/Agarose solution.
- §Cast the gel.
- §Load samples and run the gels according to the instructions in your standard protocol.
- §Use EtBr filter to image the gels.
- 4.Why GelGreen is better than the other green nucleic acid stains – SYBRGreen and SYBR Safe?
- GelGreen is a green fluorescent nucleic acid dye with UV absorption between 250 nm and 300 nm and absorption peak around 500 nm. This makes GelGreen compatible with UV transilluminator, 254 nm or a gel reader equipped with visible light excitation. Several independent laboratories tested this dye and declared its many advantages as:
- Higher sensitivity than SYBR Safe
- Higher hydrolytic and thermal stability than SYBR dyes
- Non-toxicity and non-mutagenicity unlike EtBr and the reportedly mutagenic SYBR Green
- Visualization with blue light boxes or UV light box using SYBR Green detection settings
- Safe for users and the environment than SYBR Safe and gel stains
- Compatibility with downstream gel purification, restriction digest, sequencing and cloning
- 5.How to use GelGreen for nucleic acid staining?
The post staining protocol includes the following steps:
- Run gels according to the standard protocols used in the lab;
- Put the gel in a container, for example: polypropylene staining tray and then add a GelGreen 3X staining solution in a quantity enough to submerge the gel.
- The gel should be agitated at room temperature for ~30 minutes.
- De-staining is not required but the gel can be washed in water if it is necessary.
- Image the gels with a blue light transilluminator (recommended) or a UV
The Precast gel staining, the end user should:
- Dilute GelGreen to 1X concentration in a concentrated electrophoresis buffer.
- Then add agarose powder and heat to dissolve. The GelGreen/agarose solution should be mixed thoroughly.
- Cast the gel.
- Load samples and run the gels according to the standard protocol.
- Image the gels with a blue light transilluminator or a UV transilluminator.