How does ethidium bromide work in gel electrophoresis

Allow the agarose to set at room temperature. Remove the comb and place the gel in the gel box. Gel loading dye is typically made at 6X concentration 0. Loading dye helps to track how far your DNA sample has traveled, and also allows the sample to sink into the gel. Add enough running buffer to cover the surface of the gel.

It is important to use the same running buffer as the one used to prepare the gel. Attach the leads of the gel box to the power supply. Turn on the power supply and verify that both gel box and power supply are working. Remove the lid. Slowly and carefully load the DNA sample s into the gel Fig. An appropriate DNA size marker should always be loaded along with experimental samples.

Replace the lid to the gel box. The cathode black leads should be closer the wells than the anode red leads. Double check that the electrodes are plugged into the correct slots in the power supply.

Turn on the power. Run the gel until the dye has migrated to an appropriate distance. Observing Separated DNA fragments When electrophoresis has completed, turn off the power supply and remove the lid of the gel box. Remove gel from the gel box.

Drain off excess buffer from the surface of the gel. Place the gel tray on paper towels to absorb any extra running buffer. Remove the gel from the gel tray and expose the gel to uv light. This is most commonly done using a gel documentation system Fig. DNA bands should show up as orange fluorescent bands. Take a picture of the gel Fig. Properly dispose of the gel and running buffer per institution regulations.

Representative Results Figure 5 represents a typical result after agarose gel electrophoresis of PCR products. Play Video.

Cite this Article Lee, P. Before you can use the favorites feature you must sign in or create an account. Continue with Shibboleth or Forgot Password? Please enter your email address so we may send you a link to reset your password.

Please enter your institutional email to check if you have access to this content. Please create an account to get access. Forgot Password? To request a trial, please fill out the form below. It was determined that the change in frictional coefficients brought about by the addition of EtBr is directly proportional to the fraction of base pairs of a fragment bound to EtBr. This change in friction is greatest in the largest fragments, which suggests that the stiffening of the molecule by the EtBr binding is the cause for the decreased mobility.

Specifically formulated to be a less hazardous alternative to ethidium bromide that can be used with either blue-light or UV excitation. Supplied as either a concentrate or a ready-to-use solution that can be used like an ethidium bromide solution. The stain is also suitable for staining RNA in gels.

GelRed and GelGreen from Biotium offer sensitivity, cell membrane impermeability for true safety, extreme heat and light stability as well compatibility with all downstream manipulations.

Eye care: If EtBr comes in contact with the eyes, immediately flush them with copious amounts of cold or cool water for at least 15 minutes, preferably in an emergency eyewash. Skin care: In the event of skin exposure, remove contaminated clothing and immediately wash the affected area with soap and copious amounts of cold or cool water for 15 minutes.

If swallowed or inhaled: In the case of ingestion obtain medical attention immediately. If EtBr dust is inhaled move to a source of fresh air. Spills: All labs should have a Spill Kit available and anyone who uses it must first be trained.

Spills of ethidium bromide solutions should be absorbed and the area decontaminated with soap and water. Avoid raising dust when cleaning up solid spills by mixing with water and then absorbing the solution.

All spill cleanup materials and absorbents should be bagged or placed in a sealed container with a hazardous waste label. Such gels will have an area of high background where the ethidium has not yet migrated out of the gel. They are, however, sufficient for many purposes, and do not generate as much ethidium waste Your safety office will know what level of ethidium causes a buffer to be declared a hazard.

Sensitivity is the same as Method I, and may require destaining in water or 1mM MgSO 4 to achieve the best sensitivity. In this method, bands become visible from the top and bottom of the gel as the dye diffuses into the matrix. High contrast results can often be achieved without destaining by soaking the gel until the top and bottom of the bands appear, and then leaving the gel to stand out of the staining solution for minutes.

During this time the stain will continue to diffuse into the gel, binding to the DNA at the expense of free dye. The result is a lower background without destaining. Always use plastic wrap under ethidium stained gels to avoid solarization damage to the surface of the transilluminator.

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