Lidstrom: Agarose Gel Electrophoresis

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• handy link: gel electrophoresis
  • click the gel electrophoresis button on the left

Imaging

Imaging systems

We have two imaging systems:

(1) ______ on the island

(2) Across from the SDS-PAGE gel pouring room

• Login info is taped to the monitor
• Software = GeneSnap
  • Manual

Instructions:

1. Click the green circle on the top left. This turns the camera on
2. Set the filter button (button = red/blue/green circles within a circle) to EtBr/UV
3. Turn on the light by selecting transillumination (not Et. Br) from the drop-down menu
4. Select a time. 500 ms works.
5. the buttons below the circle (now red) are:
   1. aperture (exposure time; donut-looking button)
   2. zoom (magnifying glass)
   3. focus (eyeball)
6. Click the red circle to take the picture
7. There are buttons to invert the colors (yin-yang) and sharpen (knife).

Imaging tips

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

Tips

• MassRuler is a great ladder. Use ~4uL.
  • Item # SM0403 from Thermo Scientific. Stable at RT for many months.
• 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

• You don't need much DNA. 50 ng should be plenty.

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