Lidstrom: Agarose Gel Electrophoresis: Difference between revisions
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(New page: Return to Protocols *handy link: [http://www.synbio.org.uk/dna-assembly/guidetogibsonassembly.html synbio.org/uk gel electrophoresis]) |
(→Tips) |
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Return to [[Lidstrom:Protocols|Protocols]] | Return to [[Lidstrom:Protocols|Protocols]] | ||
*handy link: [http://www.synbio.org.uk/dna-assembly/guidetogibsonassembly.html | *handy link: [http://www.synbio.org.uk/dna-assembly/guidetogibsonassembly.html gel electrophoresis] | ||
** click the gel electrophoresis button on the left | |||
== 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. | |||
[[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 09:10, 13 June 2013
Return to Protocols
- handy link: gel electrophoresis
- click the gel electrophoresis button on the left
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.
- 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.