Agarose gel electrophoresis
From OpenWetWareJump to navigationJump to search
|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.
- Cast a gel
- Place it in gel box in running buffer
- Load samples
- Run the gel
- Image the gel
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|
- Measure out the appropriate mass of agarose into a beaker with the appropriate volume of buffer (see below). The volume required depends on your gelbox / casting system -- 50mL makes a good, thick gel for a 7x10cm gelbox.
- 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.
- 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.
- 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.
- 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.
- 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|
for recipes see: Agarose gel loading dye
- 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.
- Unless your gel box is made from temperature sensitive materials, I've found the cooling step unnecessary, and risky if you get distracted easily.
- Often, the amount of dye you use can be dramatically reduced if you add it directly to your DNA sample and not to the entire gel. This is the case with SYBR green.
- Endy:Agarose gel
- Knight:Agarose gel electrophoresis
- Alm:Agarose gel electrophoresis
- Richard Lab:Agarose Gel Electrophoresis
- Wikipedia entry on agarose gel electrophoresis
- Colorado state has an excellent page with detailed instructions and photos
- Roche's Lab FAQ section on DNA work
- Methodbook.net has a nice primer on agarose gel electrophoresis
- CSH Protocols (~Molecular Cloning) protocol on agarose gel electrophoresis but restricted access