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Team:Washington/Protocols/gel electrophoresis
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=Agarose Gel Electrophoresis= | =Agarose Gel Electrophoresis= | ||
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== General Procedure == | == General Procedure == | ||
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#Run the gel | #Run the gel | ||
#Image the gel | #Image the gel | ||
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== Casting Gels == | == Casting Gels == | ||
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| align="center"|1.5 || align="center"|0.2 - 3 | | align="center"|1.5 || align="center"|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). | # 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. | # 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 | + | # '''From here on, a heat protective glove should be used any time the heated flask must be touched!''' |
| - | # Pour the agarose solution into the | + | # Let the agarose cool on the bench for ~5 minutes. |
| + | # At this point add your DNA visualization reagent, e.g., ethidium bromide at a volume appropriate for the amount of melted agarose. This amount will depend on the concentration of the stock solution of the stain. | ||
| + | # The flask will cool unevenly (surface first), so you must be careful not to cause ripples and bubbles, and should therefore be periodically swirled while the agarose is cooling. | ||
| + | # Pour the agarose solution into the gel casting apparatus. A pipette tip should be used to pop or shove to the side any bubbles. | ||
| + | # After bubbles have been popped, the comb should be inserted, and the gel should be allowed to cool for about 30 minutes, until solid. | ||
| + | # Because removal of the comb too early can cause the wells to collapse on themselves, one should always poor a gel prior to prepping the samples. | ||
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| + | Adapted thanks to [http://openwetware.org/wiki/Agarose_gel_electrophoresis OpenWetWare protocols]. | ||
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| - | + | '''← [[Team:Washington/Protocols|Back to Protocols]]''' | |
Latest revision as of 23:15, 28 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 |
- 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.
- From here on, a heat protective glove should be used any time the heated flask must be touched!
- Let the agarose cool on the bench for ~5 minutes.
- At this point add your DNA visualization reagent, e.g., ethidium bromide at a volume appropriate for the amount of melted agarose. This amount will depend on the concentration of the stock solution of the stain.
- The flask will cool unevenly (surface first), so you must be careful not to cause ripples and bubbles, and should therefore be periodically swirled while the agarose is cooling.
- Pour the agarose solution into the gel casting apparatus. A pipette tip should be used to pop or shove to the side any bubbles.
- After bubbles have been popped, the comb should be inserted, and the gel should be allowed to cool for about 30 minutes, until solid.
- Because removal of the comb too early can cause the wells to collapse on themselves, one should always poor a gel prior to prepping the samples.
Adapted thanks to [http://openwetware.org/wiki/Agarose_gel_electrophoresis OpenWetWare protocols].
