Lidstrom: Agarose Gel Electrophoresis

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Revision as of 15:14, 27 January 2014 by Janet B. Matsen (Talk | contribs)
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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.

OLD: Ethidium bromide instructions

If 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
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