Agarose gel electrophoresis

(Difference between revisions)
 Revision as of 10:25, 3 June 2009 (view source)m (XC size correction)← Previous diff Revision as of 18:29, 9 April 2010 (view source) (Added wiki link to Common Issues)Next diff → (12 intermediate revisions not shown.) Line 11: Line 11: == Casting Gels == == Casting Gels == - The amount of agarose to use in your gel depends on the DNA in question.  As a rough guide: + [[Image:Agarose gel dyes.jpg|thumb|right|175px|0.7% agarose gel with 1kbp ladder in UV and white light showing different dyes]] - {| +
- | Agarose (g/100mL) + '''The amount of agarose to use in your gel depends on the DNA in question.  Use the following table as a rough guide:''' - | DNA resolution (/kb) + - |- + {| border="1" cellpadding="5" cellspacing="0" align="center" - | 0.5 + |+ - | 1-30 + ! Agarose Concentration (g/100mL) !! Optimal DNA Resolution (kb) - |- + |-= - | 0.7 + | align="center"|0.5 || align="center"|1 - 30 - | 0.8-12 + |- - |- + | align="center"|0.7 || align="center"|0.8 - 12 - | 1.0 + |- - | 0.5-10 + | align="center"|1.0 || align="center"|0.5 - 10 - |- + |- - | 1.2 + | align="center"|1.2 || align="center"|0.4 - 7 - | 0.4-7 + |- - |- + | align="center"|1.5 || align="center"|0.2 - 3 - | 1.5 + |} - | 0.2-3 +
- |} + # 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). # 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). Line 74: Line 73: * [[Knight:Agarose gel electrophoresis]] * [[Knight:Agarose gel electrophoresis]] * [[Alm:Agarose gel electrophoresis]] * [[Alm:Agarose gel electrophoresis]] + * [[Richard Lab:Agarose Gel Electrophoresis]] + [[Agarose_gel_electrophoresis/Common_issues|Common Issues]] ==External links== ==External links==

Revision as of 18:29, 9 April 2010

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

1. Cast a gel
2. Place it in gel box in running buffer
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

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.

 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