Lidstrom: Agarose Gel Electrophoresis

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** click the gel electrophoresis button on the left
** 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.
+
-
* 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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== Imaging ==
== 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. 
 
 +
=== Imaging systems ===
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We have two imaging systems:
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 +
'''(1) ______ on the island'''
 +
 +
'''(2) Across from the SDS-PAGE gel pouring room'''
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* Login info is taped to the monitor
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* Software = GeneSnap
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** [http://sydney.edu.au/medicine/bosch/facilities/molecular-biology/digital-imaging/gbox_bioImaging_systems_user_manual.pdf Manual]
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Instructions:
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# Click the green circle on the top left.  This turns the camera on
 +
# Set the filter button (button = red/blue/green circles within a circle) to EtBr/UV
 +
# Turn on the light by selecting transillumination (not Et. Br) from the drop-down menu
 +
# Select a time.  500 ms works.
 +
# the buttons below the circle (now red) are:
 +
## aperture (exposure time; donut-looking button)
 +
## zoom (magnifying glass)
 +
## focus (eyeball)
 +
# Click the red circle to take the picture
 +
# 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.
== Tips ==
== Tips ==
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* You don't need much DNA.  50 ng should be plenty.  
* 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]]
[[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.
 +
 +
== 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

Revision as of 17:15, 27 January 2014

Return to Protocols


Contents

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

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.

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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