Gel electrophoresis is the standard laboratory procedure
for separating DNA by size for visualization and purification.
Electrophoresis uses an electrical field
to move the negatively charged DNA
through an agarose gel matrix toward a positive electrode.
Shorter DNA fragments, called bands,
migrate through the gel more quickly than longer ones.
You can determine the approximate length of a DNA fragment by running it
on an agarose gel alongside a DNA ladder,
which is a collection of DNA fragments of known lengths.
In this video, we'll show you how to prepare an agarose gel
using powered agarose and buffer.
Then we'll show you how to load your samples into the gel
and perform electrophoresis to successfully separate your DNA bands.
We'll also give you some tips and tricks to get great looking gels every time.
The first step in preparing an agarose gel is deciding what percentage of agarose to use
Agarose gels are commonly prepared in concentrations between 0.7% and 2.0%.
depending on the size of the bands that need to be separated.
The percentage of agarose in the gel solution determines the size of pores present in the gel matrix.
Which determines how quickly the DNA migrates down the length of the gel.
To resolve smaller bands from each other increase
the agarose concentration to reduce the size of the pores
the DNA is traveling through.
To resolve larger bands, lower the percentage of agarose
to increase the size of the pores.
We will be preparing a one percent gel,
which means we'll need to measure 1.5 grams of agarose,
and add it to 150 milliliters of 1x tris-acetate-EDTA buffer called TAE buffer.
You can also use another buffer: tris-borate-EDTA, also known as TBE buffer.
TBE buffer is less prone to overheating than TAE, and is better for longer electrophoresis runs.
which are often necessary to separate smaller bands of DNA.
When using TBE to prepare the gel, be sure to fill the gel box with TBE too.
Always fill the gel box with the same buffer that you used to prepare the gel.
To melt the agarose, microwave the solution for 1-3 minutes until it is completely dissolved.
Try not to over boil the solution, as some buffer will evaporate,
and will alter the final percentage of the agarose in the gel.
Microwave the gel in pulses, swirling the flask occasionally as the solution heats up.
Be careful when handling the flask as the solution will be hot.
Also be careful when swirling the flask as eruptive boiling can occur.
After microwaving the agarose solution, wait until it cools down
to 55 degrees celsius. If the flask is cool enough for you to touch
with just your gloved hand, then it's ready to pour.
To reduce time, you can add ethidium bromide directly to the gel solution,
with a final concentrate between 0.2 and 0.5 micrograms per milliliter.
We're adding 5 microliters of our lab stock to 150 milliliters of agarose gel.
Remember, ethidium bromide is a known mutagen.
Wear a lab coat, eye protection, and gloves when working with this chemical.
Once the gel has cooled, place a comb corresponding to the number of
needed wells into the gel tray, and slowly pour the gel.
Make sure the tray is properly sealed around the outside
so the gel doesn't pour out.
Use a pipette tip to push away any bubbles in the solution.
Let the newly poured gel sit at room temperate for up to 1 hour until completely solid,
and then fill the gel electrophoresis chamber with the same buffer you used to prepare the gel.
Pour until the gel is covered, and make sure that the buffer enters the wells.
Remember to load the DNA ladder next to your samples
and write down what sample went into what well using the ladder as a reference .
If you don't, your results may be very difficult to interpret.
After the samples are loaded into the wells of the gel,
connect the power source to the gel box and turn on the power.
Remember, black is negative and red is positive always run to red.
Most gels are run between 80-150 volts.
Running at a higher voltage will make the bands migrate faster,
but can also cause the gel to melt if the voltage is too high.
Keep running the gel until the visible dye is 75 percent of the way down,
which indicates you have good separation of your bands.
This usually takes between 1 to 1.5 hours,
depending on the size of your gel and the percentage of agarose that you used.
Once your gel has finished running, turn off the power and disconnect the electrodes from the power source.
Visualize your bands using ultraviolet light.
Be careful when handling the gel, as it is fragile and can easily break.
If you may be exposed to UV light, protect your skin by wearing safety goggles or a face shield.
There are a few ways to increase the resolution or crispness of your DNA bands.
You can use a gel comb that has wider or thinner teeth,
you can run the gel for a longer period of time at lower voltage,
or you can load less DNA into the well.
We usually load between 100-150 nanograms of DNA in each well,
and run the gel at 100V for 1.5 hours to get the best looking bands for most of our samples.
Gel electrophoresis is one of the fastest and least expensive ways
to confirm the size of your DNA product.
By using our tips and tricks, you will be able to get a great looking gel
with clear crisp bands to help you verify your samples every time.
Thank you for watching Addgene's electrophoresis protocol video.
Check out our other protocol pages at Addgene.org/protocols
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