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CSS/HRT 451
Wells
Guo
-qing Song And David Douches
Guo-qing
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OUTLINE
Concept
Applications
Factors affecting Migration
1. DNA or RNA Molecular Weight
2. Voltage
3. Agarose
4. Buffer
5. Visualisation
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Concept
Sambrook J, Russel DW (2001). Molecular Cloning: A Laboratory Manual 3rd Ed. Cold
Spring Harbor Laboratory Press. Cold Spring Harbor, NY
Application
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After
Before
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Southern Hybridization
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Northern Analysis
RNA
Hybridization
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2. Voltage
The higher the voltage, the faster the DNA moves. But voltage is
limited by the fact that it heats and ultimately causes the gel to melt.
High voltages also decrease the resolution (above about 5 to 8 V/cm)
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3. Agarose
Agarose gel electrophoresis can be used for the separation of DNA fragments
ranging from 50 base pair to several megabases (millions of bases) using
specialized apparatus. Increasing the agarose concentration of a gel reduces
the migration speed and enables separation of smaller DNA molecules.
The distance between DNA bands of a given length is determined by the percent
agarose in the gel. In general lower concentrations of agarose are better
for larger molecules because they result in greater separation between
bands that are close in size. The disadvantage of higher concentrations is
the long run times (sometimes days). Instead high percentage agarose gels
should be run with a pulsed field electrophoresis (PFE), or field inversion
electrophoresis.
Most agarose gels:
1. 1% gels are common for many applications.
2. 0.7%: good separation or resolution of large 510kb DNA fragments
3: 2% good resolution for small 0.21kb fragments.
4: Up to 3% can be used for separating very tiny fragments but a vertical
polyacrylamide gel is more appropriate in this case.
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4. Buffer
Brody JR, Calhoun ES, Gallmeier E, Creavalle TD, Kern SE (2004). Biotechniques. 37:598602.
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5. Visualisation
The most common dye used to make DNA or
RNA bands visible for agarose gel
electrophoresis is ethidium bromide,
usually abbreviated as EtBr. It fluoresces
under UV light when intercalated into
DNA (or RNA). By running DNA through
an EtBr-treated gel and visualizing it with
UV light, any band containing more than
~20ng DNA becomes distinctly visible.
EtBr is a known carcinogen, however, and
safer alternatives are available.
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