Team:WashU/Protocols/gel electrophoresis

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(Created page with "=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 ...")
 
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<!--Thanks to UW iGEM '11 for the above instructions.  The original source can be found here: https://2011.igem.org/Team:Washington/Protocols/gel_electrophoresis-->
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=Agarose Gel Electrophoresis=
=Agarose Gel Electrophoresis=
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== General Procedure ==
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== Making the Gel ==
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#Cast a gel
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#Weigh out the appropriate amount of agarose into a 250 mL conical flask. (For 1% gels, use 0.5g of agarose.) Add 50 mL of 0.5xTBE , swirl to mix
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#Place it in gel box in running buffer
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#Microwave for about 1 minute to dissolve the agarose (stop it after 45 seconds and give it a swirl is generally a good idea
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#Load samples
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#Leave it to cool on the bench for 5 minutes down to about 60°C (just too hot to keep holding in bare hands).
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#Run the gel
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#Add 1 µL of ethidium bromide (10 mg/mL) and swirl to mix
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#Image the gel
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#Pour the gel slowly into the tank. Push any bubbles away to the side using a disposable tip.
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#Insert the comb and double check that it is correctly positioned.
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#Leave to set for at least 30 minutes, preferably 1 hour, with the lid on if possible.
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== Casting Gels ==
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#Pour 0.5x TBE buffer into the gel tank to submerge the gel to 2–5 mm depth. This is the running buffer
'''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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# 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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== Preparing the samples ==
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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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# '''From here on, a heat protective glove should be used any time the heated flask must be touched!'''
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# Let the agarose cool on the bench for ~5 minutes.
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#Transfer an appropriate amount of each sample to a fresh microfuge tube.
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# 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.
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#Add an appropriate amount of loading buffer into each tube and leave the tip in the tube.  
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# 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.
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#Load the first well with marker.
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# Pour the agarose solution into the gel casting apparatus.  A pipette tip should be used to pop or shove to the side any bubbles.
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#Continue loading the samples and finish of with a final lane of marker
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# After bubbles have been popped, the comb should be inserted, and the gel should be allowed to cool for about 30 minutes, until solid.
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#Close the gel tank, switch on the power-source and run the gel at 5 V/cm. (a good starting point)
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# 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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#Stop the gel when the bromophenol blue has run 3/4 the length of the gel.
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#Switch off and unplug the gel tank and carry the gel (in its holder if possible) to the dark-room to look at on the UV light-box.
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<div align="center">
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<font size ="5">
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[https://2012.igem.org/Team:WashU/Protocols Back to Protocols]

Latest revision as of 21:30, 9 August 2012


Agarose Gel Electrophoresis

Making the Gel

  1. Weigh out the appropriate amount of agarose into a 250 mL conical flask. (For 1% gels, use 0.5g of agarose.) Add 50 mL of 0.5xTBE , swirl to mix
  2. Microwave for about 1 minute to dissolve the agarose (stop it after 45 seconds and give it a swirl is generally a good idea
  3. Leave it to cool on the bench for 5 minutes down to about 60°C (just too hot to keep holding in bare hands).
  4. Add 1 µL of ethidium bromide (10 mg/mL) and swirl to mix
  5. Pour the gel slowly into the tank. Push any bubbles away to the side using a disposable tip.
  6. Insert the comb and double check that it is correctly positioned.
  7. Leave to set for at least 30 minutes, preferably 1 hour, with the lid on if possible.
  8. Pour 0.5x TBE buffer into the gel tank to submerge the gel to 2–5 mm depth. This is the running buffer

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


Preparing the samples

  1. Transfer an appropriate amount of each sample to a fresh microfuge tube.
  2. Add an appropriate amount of loading buffer into each tube and leave the tip in the tube.
  3. Load the first well with marker.
  4. Continue loading the samples and finish of with a final lane of marker
  5. Close the gel tank, switch on the power-source and run the gel at 5 V/cm. (a good starting point)
  6. Stop the gel when the bromophenol blue has run 3/4 the length of the gel.
  7. Switch off and unplug the gel tank and carry the gel (in its holder if possible) to the dark-room to look at on the UV light-box.
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