Agarose gel electrophoresis

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{{back to protocols}}
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.
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.
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#Run the gel
#Run the gel
#Image the gel
#Image the gel
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== Casting Gels ==
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[[Image:Agarose gel dyes.jpg|thumb|right|175px|0.7% agarose gel with 1kbp ladder in UV and white light showing different dyes]]
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<center>
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'''The amount of agarose to use in your gel depends on the DNA in question.  Use the following table as a rough guide:'''
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{| border="1" cellpadding="5" cellspacing="0" align="center"
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|+
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! Agarose Concentration (g/100mL) !! Optimal DNA Resolution (kb)
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|-=
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| align="center"|0.5 || align="center"|1 - 30
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|-
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| align="center"|0.7 || align="center"|0.8 - 12
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|-
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| align="center"|1.0 || align="center"|0.5 - 10
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|-
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| align="center"|1.2 || align="center"|0.4 - 7
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|-
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| align="center"|1.5 || align="center"|0.2 - 3
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|}
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</center>
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# Measure out the appropriate mass of agarose into a beaker with the appropriate volume of buffer (see the documentation for your gelbox -- 50mL makes a good, thick gel for a 7x10cm gelbox). 
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# Microwave until the agarose is fully melted.  This depends strongly on your microwave, but a 90 seconds at full power or 3 minutes at half power seem to provide decent results.  As long as you do not burn the agarose and nothing bubbles over, this step is robust.
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# Let the agarose cool on your bench until touching the bottom of the beaker with your bare hand doesn't burn you (~5 minutes for a 50mL gel).  At this point add your DNA stain, e.g., ethidium bromide.  The beaker will cool unevenly (surface first), so you must be careful not to cause ripples and bubbles.
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# While the solution is cooling, seal the open edges of your gel box with one long piece of masking tape on each side.  Make sure it is sealed well or the gel will leak.
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# Pour the agarose solution into the taped gelbox.  Carefuly pop or shove to the side any bubbles, put in the comb, and let it cool for about 30 minutes, until the gel is solid.
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If your gel is at all purple, and you are using ethidium bromide as the DNA stain, you need to decrease your concentration by at least a factor of ten.
== 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;
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* [[SB]] - Sodium borate - low heat generation with good resolution. Allows for higher voltage (250-200V) electrophoresis without melting.
== Loading dyes ==
== Loading dyes ==
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  |-
  |-
  | xylene cyanol
  | xylene cyanol
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  | 4000-5000 bp
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  | 10'000-5000 bp
  | 750 bp
  | 750 bp
  |-
  |-
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<nowiki>*</nowiki>sieving agarose
<nowiki>*</nowiki>sieving agarose
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see for recipes: [[Agarose gel loading dye]]
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for recipes see: [[Agarose gel loading dye]]
==Notes==
==Notes==
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* [[Endy:Agarose gel]]
* [[Endy:Agarose gel]]
* [[Knight:Agarose gel electrophoresis]]
* [[Knight:Agarose gel electrophoresis]]
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* [[Alm:Agarose gel electrophoresis]]
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* [[Richard Lab:Agarose Gel Electrophoresis]]
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[[Agarose_gel_electrophoresis/Common_issues|Common Issues]]
==External links==
==External links==
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* Wikipedia entry on [http://en.wikipedia.org/wiki/Agarose_gel_electrophoresis agarose gel electrophoresis]
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* Wikipedia entry on [http://en.wikipedia.org/wiki/Agarose_gel_electrophoresis agarose gel electrophoresis] [[Image:3stars.png]]
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* Colorado state has an excellent [http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/agardna.html page with detailed instructions and photos] [[Image:3stars.png]]
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* [https://www.roche-applied-science.com/PROD_INF/MANUALS/labfaqs/lab_faqs_%2001.pdf Roche's Lab FAQ section on DNA work] [[Image:3stars.png]]
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* Methodbook.net has a nice [http://www.methodbook.net/dna/agarogel.html primer on agarose gel electrophoresis] [[Image:2stars.png]]
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* CSH Protocols (~Molecular Cloning) protocol on [http://www.cshprotocols.org/cgi/content/full/2006/2/pdb.prot4020 agarose gel electrophoresis] [[Image:3stars.png]] but restricted access
[[Category:Protocol]]
[[Category:Protocol]]
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[[Category:In vitro]]
[[Category:DNA]]
[[Category:DNA]]
-
[[Category:In vitro]]
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[[Category:RNA]]

Revision as of 17:29, 9 April 2010

back to protocols

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.

Contents

General Procedure

  1. Cast a gel
  2. Place it in gel box in running buffer
  3. Load samples
  4. Run the gel
  5. Image the gel

Casting Gels

0.7% agarose gel with 1kbp ladder in UV and white light showing different dyes
0.7% agarose gel with 1kbp ladder in UV and white light showing different dyes

The amount of agarose to use in your gel depends on the DNA in question. Use the following table as a rough guide:

Agarose Concentration (g/100mL) Optimal DNA Resolution (kb)
0.5 1 - 30
0.7 0.8 - 12
1.0 0.5 - 10
1.2 0.4 - 7
1.5 0.2 - 3
  1. Measure out the appropriate mass of agarose into a beaker with the appropriate volume of buffer (see the documentation for your gelbox -- 50mL makes a good, thick gel for a 7x10cm gelbox).
  2. Microwave until the agarose is fully melted. This depends strongly on your microwave, but a 90 seconds at full power or 3 minutes at half power seem to provide decent results. As long as you do not burn the agarose and nothing bubbles over, this step is robust.
  3. Let the agarose cool on your bench until touching the bottom of the beaker with your bare hand doesn't burn you (~5 minutes for a 50mL gel). At this point add your DNA stain, e.g., ethidium bromide. The beaker will cool unevenly (surface first), so you must be careful not to cause ripples and bubbles.
  4. While the solution is cooling, seal the open edges of your gel box with one long piece of masking tape on each side. Make sure it is sealed well or the gel will leak.
  5. Pour the agarose solution into the taped gelbox. Carefuly pop or shove to the side any bubbles, put in the comb, and let it cool for about 30 minutes, until the gel is solid.

If your gel is at all purple, and you are using ethidium bromide as the DNA stain, you need to decrease your concentration by at least a factor of ten.

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;
  • SB - Sodium borate - low heat generation with good resolution. Allows for higher voltage (250-200V) electrophoresis without melting.

Loading dyes

dye 0.5-1.5% agarose 2.0-3.0% agarose*
xylene cyanol 10'000-5000 bp 750 bp
bromophenol blue 400-500 bp 100 bp

*sieving agarose

for recipes see: Agarose gel loading dye

Notes

See also

Specific Protocols

Common Issues

External links

Personal tools