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Team:Washington/Protocols/gel electrophoresis
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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:''' | '''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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# 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. | # 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. | # 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. | ||
| - | + | # Pour the agarose solution into the 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. | |
| - | # Pour the agarose solution into the | + | |
| - | + | Adapted with many thanks from [http://openwetware.org/wiki/Agarose_gel_electrophoresis OpenWetWare protocols]. | |
Revision as of 01:24, 14 September 2011
Agarose Gel Electrophoresis
General Procedure
- Cast a gel
- Place it in gel box in running buffer
- Load samples
- Run the gel
- Image the gel
Casting Gels
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 |
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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).
- 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.
- Pour the agarose solution into the 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.
Adapted with many thanks from [http://openwetware.org/wiki/Agarose_gel_electrophoresis OpenWetWare protocols].
