Description
Description
The most commonly used stain for
detecting DNA/RNA is Ethidium Bromide (EtBr). EtBr is a DNA
intercalator, inserting itself into the spaces between the base
pairs of the double helix. EtBr possesses UV absorbance maxima at
**0 and **0 nm. Additionally, it can absorb energy from nucleotides
excited by absorbance of **0 nm radiation. Ethidium re-emits this
energy as yellow/orange light centered at **0 nm. The fluorescence
of EtBr in aqueous solution is significantly lower than that of the
intercalated dye. Ethidium Bromide Solution, Molecular Biology
Grade (*0mg/ml), is a fluorescent dye suitable for staining nucleic
acids after electrophoresis or in cesium chloride gradients. The
solution can be used to detect both double-stranded and
single-stranded DNA.
Quality
Control:
Each lot of Ethidium Bromide
Solution is tested and certified to be free of DNase, RNase and
protease activity.
Storage
Conditions
Store at *2–*5°C.
Detection of DNA/RNA using
Ethidium Bromide
CAUTION:Ethidium Bromide Eis a potent
mutagen. Handle only with gloves and proper
precautions
Protocol:（add 5µl EtBr to**0ml agarose
gel solution）
1. Prepare
**0 ml of agarose gel solution (concentration from 0.**2.0%) in a
**0 ml flask and mix it thoroughly. Place the flask in the
microware, heat on high until the solution is completely clear and
no small floating particles are visible (about **3
minutes).
2. Add
5µl of EtBr to the gel solution. Swirl the flask gently to mix the
solution and avoid forming bubbles.
3. While
the gel solution cools below ****0℃, pour it into the gel tray
until the comb teeth are immersed about 1/**1/2 into the gel
solution.
4. Allow
the agarose gel to cool until solidified. Load samples on the gel
and perform electrophoresis.
5. Detect
the bands under UV illumination.
Method II - Post
Run Staining
1. Prepare enough 0.5µg/ml EtBr in
water or buffer to completely submerge the gel. This solution is
stable for **2 months at room temperature in the dark.
2. After the run submerge the gel in
the staining solution for ****0 minutes (depending upon gel
thickness).
3. Place the gel on plastic wrap on a
UV light box and observe under **0nm illumination. Bands will
appear bright orange on a pale orange background.
Notes:
(1) This protocol minimizes the amount of EtBr waste created with
each gel run.
(2) Sensitivity is the same as method
I, and may require destaining in water or 1mM
MgSO4
to achieve the best
sensitivity.
(3) 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 ****0 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.
(4) Always use plastic wrap under
Ethidium stained gels, to avoid solarization damage to the surface
of the transilluminator.
