Lidstrom: Agarose Gel Electrophoresis: Difference between revisions
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** click the gel electrophoresis button on the left | ** click the gel electrophoresis button on the left | ||
* add | |||
== 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 | |||
** [http://sydney.edu.au/medicine/bosch/facilities/molecular-biology/digital-imaging/gbox_bioImaging_systems_user_manual.pdf Manual] | |||
Instructions: | |||
# 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 == | |||
* 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. | |||
== 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 15:15, 27 January 2014
Return to Protocols
- 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
Instructions:
- 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
- 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.
- You don't need much DNA. 50 ng should be plenty.
- 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