Lidstrom: Agarose Gel Electrophoresis

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== Starting with an empty box ==
== Starting with an empty box ==
* Fill with TBE until it covers the gel holding tray.
* Fill with TBE until it covers the gel holding tray.
-
* Add 40? uL of ethidium bromide if your gels don't already have it within
+
* 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.  
** If your buffer already has some ethidium bromide but it needs more, 20 uL is probably good.  
* Load sample  
* Load sample  
 +
** 1.7 uL of a PCR product is usually plenty
* Load ladder
* Load ladder
** If using MassRuler, 4 uL is good for skinny lanes.  
** If using MassRuler, 4 uL is good for skinny lanes.  
-
* Run @ 100 V for 20 min and check the gel.
+
* 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.
 +
[[image:gel sat 16 hours between photos.jpg|thumb|center|gel sat 16 hours between photos]]
 +
* You don't need much DNA.  50 ng should be plenty.
 +
[[image:75 and 150 ng DNA on a gel.jpg|thumb|center|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.

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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