Laboratory 4i
Making Agarose Gels for Separating
and Analyzing DNA Fragments
Background
Agarose gels are typically used to separate and analyze DNA molecules ranging in length
from about 500 to 25,000 base pairs (bp). The ability of a gel to separate molecules is called its
“resolving power” and is mainly determined by the concentration of agarose in the gel. Most
agarose gels have concentrations between 0.6% and 3.0% agarose in buffer.
Agarose gels are prepared by dissolving powdered agarose in a certain volume of
electrophoresis buffer. The agarose-buffer mixture has to be boiled for the agarose to go into
solution. Powdered agarose may be purchased from a chemical supply house. The buffer may
be prepared in the lab or purchased premade, usually as a concentrated stock solution (see
Figure 4.17).
The most common agarose gel electrophoresis buffer is 1X TAE (TRIS, Acetic Acid, and
EDTA). Although used for decades, the TRIS molecule is a rather poor conductor of electricity.
Advances in electrophoresis technology have found some buffering molecules that do a better job
of conducting electricity than TRIS. One excellent electrophoresis buffering molecule is lithium
borate (LB). Lithium borate conducts electric better and doesn’t generate as much heat during an
electrophoresis as TRIS does. The result is that LB buffered gels may be run 6–8X faster than TAE
gels. Now, many labs use 1X LB buffer to run faster, better gels. Read more about LB buffers at
www.fasterbettermedia.com. In this lab manual, agarose gel labs provide the option of either a
TAE-buffered gel or a LB-buffered gel system.
The concentration of agarose used in a gel depends on the type of molecules to be analyzed.
The longer the molecules in a sample, the less concentrated the gel should be. Too high a
concentration may impede the movement of molecules through a gel. The lowest practical
working concentration of an agarose gel is about 0.6%, or 0.6 g, of agarose dissolved in 100 mL of
buffer. This is used when a sample is composed mostly of long DNA fragments.
As the concentration of agarose in a gel increases, the agarose threads are pushed closer
together, making it difficult for larger molecules to move through them. A higher concentration
gel separates short DNA fragments well. Gels of 0.8% and 1% are typical for plasmid analysis.
Gels of higher concentration, up to about 3%, may be used with smaller DNA pieces. A lower
concentration gel resolves long DNA fragments, such as genomic DNA, best.
Purpose
To prepare and pour an agarose gel for DNA fragment analysis.
Boil agarose and buffer
mixture until dissolved.
Make sure
to add a
comb.
Figure 4.17. Pouring agarose gels.
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Chapter 4 Laboratory Manual
agarose dissolved in
electrophoresis buffer
(either 1X TAE or 1X LB buffer)
Cool to 65°C, then
pour a 7mm thick gel
(30 mL for most gel trays).