Lidstrom: Agarose Gel Electrophoresis

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*handy link: [http://www.synbio.org.uk/dna-assembly/guidetogibsonassembly.html gel electrophoresis]
*handy link: [http://www.synbio.org.uk/dna-assembly/guidetogibsonassembly.html gel electrophoresis]
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** click the gel electrophoresis button on the left
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== Starting with an empty box ==
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* Fill with TBE until it covers the gel holding tray.
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* Add 40 uL of ethidium bromide if your gels don't already have it mixed into the liquefied agarose.
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** If your buffer already has some ethidium bromide but it needs more, 20 uL is probably good.
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* Load sample
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** 1.7 uL of a PCR product is usually plenty
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* Load ladder
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** If using MassRuler, 4 uL is good for skinny lanes.
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* Run @ 110 V for 20 min and check the gel.
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** 40 min is pretty safe for 0.7 - 1% agarose gels
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== Imaging ==
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* Exposures should be < 0.5 seconds to minimize background emissions.  If you need longer, you probably should have had more ethidium bromide. 
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== Tips ==
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* If you have a voltage source that will stop after a set amount of time, feel free to run your gel as you leave and image it in the morning.  Diffusion of bands shouldn't be a big problem unless they are << 500 bp.
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** Don't do this if you are going to cut & gel purify the band.
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[[image:gel sat 16 hours between photos.jpg|thumb|center|gel sat 16 hours between photos]]
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* You don't need much DNA.  50 ng should be plenty.
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[[image:75 and 150 ng DNA on a gel.jpg|thumb|center|75 and 150 ng DNA on a 0.7% agarose gel]]
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* TBE buffer can be re-used for '''months'''. 
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** This is nice, because you shouldn't dump ethidium bromide containing solution down the drain.
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** Janet usually uses it heavily for 4+ months before replacing it. 
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** Always add DI water to replace evaporated water, '''not''' TBE buffer
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* Agarose gels can be "drained" and re-used several times.
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** Don't use a previously used gel for DNA you will cut and purify for cloning/sequencing.
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** Drain with 100V for ~90 min.

Revision as of 12:10, 13 June 2013

Return to Protocols

Starting with an empty box

  • Fill with TBE until it covers the gel holding tray.
  • Add 40 uL of ethidium bromide if your gels don't already have it mixed into the liquefied agarose.
    • If your buffer already has some ethidium bromide but it needs more, 20 uL is probably good.
  • Load sample
    • 1.7 uL of a PCR product is usually plenty
  • Load ladder
    • If using MassRuler, 4 uL is good for skinny lanes.
  • Run @ 110 V for 20 min and check the gel.
    • 40 min is pretty safe for 0.7 - 1% agarose gels

Imaging

  • Exposures should be < 0.5 seconds to minimize background emissions. If you need longer, you probably should have had more ethidium bromide.


Tips

  • If you have a voltage source that will stop after a set amount of time, feel free to run your gel as you leave and image it in the morning. Diffusion of bands shouldn't be a big problem unless they are << 500 bp.
    • Don't do this if you are going to cut & gel purify the band.
gel sat 16 hours between photos
gel sat 16 hours between photos
  • You don't need much DNA. 50 ng should be plenty.
75 and 150 ng DNA on a 0.7% agarose gel
75 and 150 ng DNA on a 0.7% agarose gel
  • TBE buffer can be re-used for months.
    • This is nice, because you shouldn't dump ethidium bromide containing solution down the drain.
    • Janet usually uses it heavily for 4+ months before replacing it.
    • Always add DI water to replace evaporated water, not TBE buffer
  • Agarose gels can be "drained" and re-used several times.
    • Don't use a previously used gel for DNA you will cut and purify for cloning/sequencing.
    • Drain with 100V for ~90 min.
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