Quick Guide: Visualizing DNA
Agarose gel electrophoresis is used to separate DNA fragments in complex mixtures according to their size. However, because DNA is clear and colorless these bands cannot be see with the naked eye. Edvotek® offers several different methods for visualizing the DNA separated by electrophoresis.
Fluorescent DNA Stains:Research laboratories commonly use fluorescent DNA stains because they are extremely sensitive, making it easy to quantify small amounts of DNA. In order to visualize the DNA fragments, an ultraviolet (UV) light source (such as a transilluminator) is used to excite the fluorescent molecules. We offer two fluorescent DNA stains: InstaStain® Ethidium Bromide and SYBR® Safe DNA Stain.
Visible Dye-based DNA Stains:
Although they are less sensitive than fluorescent stains, dye-based DNA stains are an excellent alternative for the teaching classroom, as they are non-toxic and require no special equipment for visualization. The molecules of the DNA stain possess a positive charge, which allows them to bind to the negatively charged backbone of DNA. The DNA fragments are easily visuzlized because the bound dye molecules stain them with an intense blue color. We offer two visible dye-based DNA stains: InstaStain® Blue and Flash Blue Stain.
Which DNA Stain Should I Use?
InStaStain® Ethidium Bromide
The most commonly used fluorescent DNA stain is Ethidium Bromide (EtBr). Individual EtBr molecules can squeeze between neighboring base pairs in a DNA double helix in a process known as "intercalation." When excited with UV light, any EtBr intercalated into the DNA fluoresces and produces a bright orange light.
However, because EtBr is a potential mutagen, it must be handled with care. InstaStain® Ethidium Bromide provides the sensitivity of EtBr while minimizing potential contact with hazardous materials by delivering a small amount of stain to the agarose gel via a special paper backing.
- 1. Carefully REMOVE the agarose gel and casting tray from the electrophoresis chamber. SLIDE the gel off of the casting tray on to a piece of plastic wrap on a flat surface. **DO NOT STAIN GELS IN THE ELECTROPHORESIS APPARATUS.**
- 2. MOISTEN the gel with a few drops of electrophoresis buffer.
- 3. Wearing gloves, REMOVE and DISCARD the clear plastic protective sheet from the unprinted side of the InstaStain® card(s). PLACE the unprinted side of the InstaStain® Ethidium Bromide card(s) on the gel. Each InstaStain® Ethidium Bromide will stain 49 cm2 of gel (7x7cm).
- 4. With a gloved hand, REMOVE air bubbles between the card and the gel by firmly funning your fingers over the entire surface. Otherwise, those regions will not stain.
- 5. PLACE the casting tray on top of the gel/card stack. PLACE a small weight (i.e. an empty glass beaker) on top of the casting tray. This ensures that the InstaStain® Ethidium Bromide card is in direct contact with the gel surface. STAIN gel for 3-5 minutes for an 0.8% gel or 8-10 minutes for a gel 1.0% or greater.
- 6. REMOVE the InstaStain® Ethidium Bromide card(s). VISUALIZE the gel using a long wavelength ultraviolet transilluminator (300 nm). DNA should appear as bright orange bands on a dark background.
Staining with InstaStain® Ethidium Bromide Video:
In-Gel SYBR® Safe DNA Staining Method (Preferred Method)
FlashBlue™ is a proprietary DNA stain that offers simple and rapid staining of agarose gels. FlashBlue™ is provided as a concentrated liquid stain that, when diluted, can be used for both rapid and overnight staining of DNA fragments.
- 1. DILUTE 10mL of 10X concentrated FlashBlue™ with 90mL of distilled water in a flask. MIX well.
- 2. REMOVE the agarose gel and casting tray from the electrophoresis chamber. SLIDE the gel off the casting tray into a small, clean gel-staining tray.
- 3. COVER the gel with the 1X FlashBlue™ stain solution. STAIN the gel for 2-3 minutes. For best results, use an orbital shaker to gently agitate the gel while staining. STAINING THE GEL FOR LONGER THAN 3 MINUTES WILL REQUIRE EXTRA DESTAINING TIME.
- 4. POUR the 1X FlashBlue™ back into the flask (the stain can be reused). COVER the gel with warm water (40-45℃). Gently RINSE the gel for 20-30 seconds. POUR off the water.
- 5. COVER the gel with clean, warm water (40-45℃). DESTAIN for 5-15 minutes with gentle shaking (longer periods will yield better results). DNA bands will start to appear after 5 minutes of destaining. Changing the water frequently will accelerate the destaining.
- 6. Carefully REMOVE the gel from the destaining liquid. VISUALIZE results using a white light visualization system. DNA will appear as dark blue bands on a light blue background.
Alternative FlashBlue™ Staining Protocol
- 1. DILUTE 1 mL of 10X FlashBlue™ stain with 149 mL distilled water.
- 2. COVER the gel with diluted FlashBlue™ stain.
- 3. SOAK the gel in the staining liquid for at least three hours. For best results, stain gels overnight.
- 4. Carefully REMOVE the gel from the staining liquid. VISUALIZE results using a white light visualization system. DNA will appear as dark blue bands on a light blue background.
Staining with FlashBlue™ Video:
InstaStain® Blue does not require the formulation, storage, and disposal of large volumes of liquid stain. Each InstaStain® Blue card contains a small amount of blue DNA stain. When the card is placed in water, the DNA stain is released. This solution simultaneously stains and destains the gel, providing uniform gel staining with minimal liquid waste and mess.
- 1. Carefully SLIDE the agarose gel from its casting tray into a small, clean tray containing at least 75ml of distilled/deionized water or used electrophoresis buffer. The gel should be completely submerged.
*Note: Appropriate staining trays include large weigh boats and small, plastic food containers.*
- 2. Gently FLOAT the InstaStain® Blue card(s) on top of the liquid with the stain (blue side) facing the gel. Each InstaStain® Blue card will stain 49 cm2 of gel (7 x 7 cm). REMOVE the InstaStain® card(s) after 30 seconds.
- 3. COVER the tray with plastic wrap to prevent evaporation. SOAK the gel in the staining liquid for at least 3 hours. The gel can remain in the liquid overnight if necessary.
- 4. Carefully REMOVE the gel from the staining tray and DOCUMENT results