Biology Reference
In-Depth Information
Figure 5.5
Photograph of DNA stained with ethidium bromide and illuminated with UV light. The
bands are a pink-purple color.
Agarose
gel is used for longer DNA molecules and
polyacrylamide
for shorter.
Nondenaturing polyacrylamide gels can be used to separate double-stranded
DNA fragments between 6bp (20% acrylamide) and 1000bp (3% acrylamide).
Nondenaturing agarose gels can separate DNA fragments between 70bp (3%
agarose) and 10,000bp (0.1% agarose). Single-stranded DNA can be separated
by agarose or polyacrylamide gel electrophoresis by including a denaturing
reagent in the gel.
DNA from 60 kb to 0.1 kb can be detected with agarose gels containing differ-
ent percentages of agarose. Agarose gels are usually electrophoresed at room
temperature, except for low-percentage agarose gels (
<
0.5%) or low-melting
temperature gels that are easier to handle at cooler temperatures.
Agarose powder comes in grades that vary in purity and melting temperature.
Type II agarose is generally used, although it contains contaminants that coelute
with DNA and inhibit most commonly used enzymes. This means that DNA must
be purified after elution from this gel if it is to be ligated or cut with restriction
enzymes. An alternative involves using a high-quality, low-melting temperature aga-
rose that melts at 65°C and sets at 30°C. Low-melt agarose allows DNA to remain
double-stranded and also allows many enzymes to be used in the liquid agar.
Polyacrylamide gels result from polymerization of acrylamide monomers into
linear chains and the linking of these chains with
N
,
N
′
-methylenebis-acrylamide
(often called bis). The concentration of acrylamide and the ratio of acrylamide
to bis determine the pore size of the resultant three-dimensional network and
thus its sieving effect on nucleic acids. Polyacrylamide gels can be used to purify
