Agarose gel electrophoresis: Difference between revisions
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Agarose gel electrophoresis is used to separate DNA or RNA molecules by size. Nucleic acids are negatively charged and are moved through an agarose matrix by an electric field (electrophoresis). Shorter molecules move faster and migrate further. | |||
== General Procedure == | == General Procedure == | ||
#Cast a gel | #Cast a gel | ||
#Place it in gel box in running buffer | #Place it in gel box in running buffer | ||
| Line 10: | Line 9: | ||
== Buffers == | == Buffers == | ||
* [[TAE]] - better resolution of fragments >4kb; | * [[TAE]] - better resolution of fragments >4kb; | ||
* [[TBE]] - better resolution of 0.1-3kb fragments; TBE is better suited for high-voltage (>150V) electrophoresis than TAE because of its higher buffering capacity and lower conductivity; | * [[TBE]] - better resolution of 0.1-3kb fragments; TBE is better suited for high-voltage (>150V) electrophoresis than TAE because of its higher buffering capacity and lower conductivity; | ||
== Loading dyes == | == Loading dyes == | ||
{| cellpadding=5 style="border: 1px solid #CC9;" | {| cellpadding=5 style="border: 1px solid #CC9;" | ||
| dye | | dye | ||
| Line 32: | Line 29: | ||
<nowiki>*</nowiki>sieving agarose | <nowiki>*</nowiki>sieving agarose | ||
see for recipes: [[Agarose gel loading dye]] | |||
[[ | |||
==Notes== | ==Notes== | ||
*If you are getting unexpected bands on your gel you may want to look at the [[Agarose gel electrophoresis/Common issues | common issues in agarose gel electrophoresis page]]. | *If you are getting unexpected bands on your gel you may want to look at the [[Agarose gel electrophoresis/Common issues | common issues in agarose gel electrophoresis page]]. | ||
*If you have no experience with gel electrophoresis or are explaining it to someone new, [http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/virgel.html here] is a cute java demo of what happens. | *If you have no experience with gel electrophoresis or are explaining it to someone new, [http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/virgel.html here] is a cute java demo of what happens. | ||
==See also== | |||
* [[Agarose gel loading dye]] | |||
Specific Protocols | |||
* [[Endy:Agarose gel]] | |||
* [[Knight:Agarose gel electrophoresis]] | |||
==External links== | |||
* Wikipedia entry on [http://en.wikipedia.org/wiki/Agarose_gel_electrophoresis agarose gel electrophoresis] | |||
Revision as of 05:05, 20 April 2007
Agarose gel electrophoresis is used to separate DNA or RNA molecules by size. Nucleic acids are negatively charged and are moved through an agarose matrix by an electric field (electrophoresis). Shorter molecules move faster and migrate further.
General Procedure
- Cast a gel
- Place it in gel box in running buffer
- Load samples
- Run the gel
- Image the gel
Buffers
- TAE - better resolution of fragments >4kb;
- TBE - better resolution of 0.1-3kb fragments; TBE is better suited for high-voltage (>150V) electrophoresis than TAE because of its higher buffering capacity and lower conductivity;
Loading dyes
| dye | 0.5-1.5% agarose | 2.0-3.0% agarose* |
| xylene cyanol | 4000-5000 bp | 750 bp |
| bromophenol blue | 400-500 bp | 100 bp |
*sieving agarose
see for recipes: Agarose gel loading dye
Notes
- If you are getting unexpected bands on your gel you may want to look at the common issues in agarose gel electrophoresis page.
- If you have no experience with gel electrophoresis or are explaining it to someone new, here is a cute java demo of what happens.
See also
Specific Protocols
External links
- Wikipedia entry on agarose gel electrophoresis
