Handouts-3-Agarose Gel Electrophoresis

Agarose Gel Electrophoresis
Background Information
Gel electrophoresis is the standard lab procedure for separating DNA by size (e.g. length in base pairs) for
visualization and purification. Electrophoresis uses an electrical field to move the negatively charged
DNA toward a positive electrode through an agarose gel matrix. The gel matrix allows shorter DNA
fragments to migrate more quickly than larger ones. Thus, you can accurately determine the length of a
DNA segment by running it on an agarose gel alongside a DNA ladder (a collection of DNA fragments of
known lengths).
Protocol: Gel Electrophoresis
Loading Samples and Running an Agarose Gel:

1. Once solidified, place the agarose gel into the gel box (electrophoresis unit).
2. Fill gel box with 1xTAE (or TBE) until the gel is covered.
Note: Rememb
er, if you
added EtBr to
your gel, add
some to the
buffer as well.
EtBr is
positively
charged and
will run the
opposite
direction from
the DNA. So if
you run the gel
without EtBr in
the buffer you
will reach a point where the DNA will be in the bottom portion of the gel, but all of the EtBr
will be in the top portion and your bands will be differentially intense. If this happens, you
can just soak the gel in EtBr solution and rinse with water to even out the staining after the
gel has been run, just as you would if you had not added EtBr to the gel in the first place.
3. Add loading buffer to each of your PCR product. We are using a 6X loading buffer, so add 10 µl
to you 50 µl PCR product.
Note: Loading buffer serves two purposes: 1) it provides a visible dye that helps with gel
loading and will also allows you to gauge how far the gel has run while you are running
your gel; and 2) it contains a high % glycerol, so after adding it your sample is heavier
than water and will settle to the bottom of the gel well, instead of diffusing in the buffer.
4. Carefully load a molecular weight ladder into the first lane of the gel.
Note: When loading the sample in the well,
maintain positive pressure on the sample to
prevent bubbles or buffer from entering the tip.
Place the very top of the tip of the pipette into the
buffer just above the well. Very slowly and
steadily, push the sample out and watch as the
sample fills the well. After all of the sample is
unloaded, push the pipettor to the second stop
and carefully raising the pipette straight out of
the buffer.
5. Carefully load 30 µl of your samples into the additional wells of the gel. Ask your instructors to
keep the other 30 µl of samples at -20C in case you still need them. When you load the samples,
you may want to skip one lane between the two samples. It will make it easier to cut the sample
out during the purification step.
6. Run the gel at 100-150V until the dye line is

approximately 70% of the way down the gel.
Note: Black is negative, red is positive. (The

DNA is negatively charged and will run towards
the positive electrode.) Always Run to Red.
Note: A typical run time is about 0.5-1 hours,

depending on the gel concentration and voltage.
7. Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the
gel from the gel box.
8. (Optional) If you did not add EtBr to the gel and buffer, place the gel into a container filled with
100 mL of TAE running buffer and 5μL of EtBr, place on a rocker for 20-30 minutes, replace EtBr
solution with water and destain for 5 minutes.
9. Using any device that has UV light, visualize your DNA fragments.
Note: When using UV light, protect your skin by wearing safety goggles or a face shield,
gloves and a lab coat.
Note: If you will be purifying the DNA for later use, use long-wavelength UV and expose
for as short a time as possible to minimize damage to the DNA.
Note: The fragments of DNA are usually referred to as ‘bands’ due to their appearance on
the gel.
Analyzing Your Gel:
Using the DNA ladder in the first lane as a guide (the manufacturer's
instruction will tell you the size of each band), you can interpret the
bands that you get in your sample lanes to determine if the resulting
DNA bands that you see are as expected or not.
Thermo Scientific GeneRuler 1 kb Plus DNA Ladder 
Purifying DNA from Your Gel:

If you are conducting certain procedures, such as molecular cloning, you will need to purify the DNA
away from the agarose gel.
Tips and FAQ

 How do you get better resolution of bands?
A few simple ways to increase the resolution (crispness) of your DNA bands include: a) running
the gel at a lower voltage for a longer period of time; b) using a wider gel comb; or c) loading less
DNA into well.
 How do you get better separation of bands?
If you have similarly sized bands that are running too close together you can adjust the agarose
percentage of the gel to get better separation. A higher percentage agarose gel will help resolve
smaller bands from each other, and a lower percentage gel will help separate larger bands.
 10% Rule:
For each sample you want to load on a gel, make 10% more volume than needed because several
microliters can be lost in pipetting. For example, if you want to load 1.0μg in 10μL, make 1.1μg in
11μL.

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Agarose Gel Electrophoresis

Protocol: Gel Electrophoresis

Loading Samples and Running an Agarose Gel:

6. Run the gel at 100-150V until the dye line is

Note: Black is negative, red is positive. (The

Note: A typical run time is about 0.5-1 hours,

Thermo Scientific GeneRuler 1 kb Plus DNA Ladder 

Purifying DNA from Your Gel:

Tips and FAQ

 How do you get better separation of bands?

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