Team:WashU/Protocols/gel electrophoresis

From 2012.igem.org

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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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== Making the Gel ==
== Making the Gel ==
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#Weigh out the appropriate amount of agarose into a 250 mL conical flask. Add 50 mL of 0.5xTBE , swirl to mix
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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
#Microwave for about 1 minute to dissolve the agarose (stop it after 45 seconds and give it a swirl is generally a good idea
#Microwave for about 1 minute to dissolve the agarose (stop it after 45 seconds and give it a swirl is generally a good idea
#Leave it to cool on the bench for 5 minutes down to about 60°C (just too hot to keep holding in bare hands).
#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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#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.
#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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==Source==
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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 University Washington gel electrophoresis protocol]
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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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