How to Prepare an Electrophoresis Argarose Gel

This instrucable illustrates the process of casting, loading, and processing an electrophoresis argarose gel. Gel electrophoresis separates biological molecules based on size and weight by utilizing electricity. Polar molecules move through the gel at different rates resulting in distinct bands. The location of these bands, relative to a standard, indicates the approximate size of the material in each sample. These samples usually consist of DNA, RNA, or protein molecules. The uses of gel electrophoresis include: estimation of the size of cloned DNA, analysis of PCR products, or separation of genomic DNA. Because chemicals used in gel electrophoresis can be hazardous, no one should attempt casting a gel without basic lab safety training. Also, anyone attempting to photograph the gel for analysis should be trained on using a UV camera. After adequately preparing stock solutions, casting a gel can take up to an hour depending on the number of samples. Don't worry though. Most of the time is spent waiting for the gel to try and process and the gel. After reading this instructable, you should not have problems performing argarose gel electrophoresis, which is a common process utilized in many biological labs.

Step 1 Gather Materials

Stock Solution Chemicals
1) 50X TAE solution
2) 1 liter plastic bottle
3) 250 milliliter flask
4) 2.5 g agarose
5) Distilled water

Casting Materials
1) Gel plate
2) Gel form
3) Comb
4) Tape
5) Microwave

Electrophoresis Equipment
1) Pipette aid
2) Pipette tips
3) Standard/Ladder
4) Samples with dye
5) Ethidium Bromide
6) Plastic pipette
7) Gel box
8) Gel box lid
8) Power supply
9) UV camera

Safety Equipment
1) Gloves 
2) Lab coat
3) Safety glasses
4) Oven mitts

NOTE : Before starting any of the procedure, make sure to wear gloves, a lab coat, and eye protection. See http://fscimage.fishersci.com/msds/45442.htm for MSDS on Ethidium Bromide.

WARNING : Ethidium Bromide is a known mutagen as stated in the MSDS. Handle with care as specified by your laboratory procedures.

Comments

[isacco]

Congratulations for this instructable.
Something more about the power supply settings. The voltage can be set up to 5 Volts per cm of distance between electrodes. Above 5 V/cm a cooling system is needed to avoid gel overheating or melting.

[Kiteman]

Yay for hands-on science!

Is it possible to record results with a visible-light camera?

[isacco]

If you use an appropriate filter, you can take pictures with any digital or film camera. The light intensity of DNA fluorescence under UV litght is quite low. As a consequence, long exposure time are usually required, therefore the camera should be mounted on a stable holder or tripod.

[wblack3]

Yep. Just need an orange filter for EB, or a green filter for SYBRgreen. Can see with plain old eyes and ordinary cameras, but not so good under UV, and can cause damage to eyes, or worse, fogs the film!

[20StoryLincoln]

That's actually how the camera setup at the lab I work in works; the system we use is pretty much a camera (albeit a super-sensitive one) in a dark box with an array of UV lights set beneath a transparent stand where the gel is placed.

Also, though I've never tried it myself, I do know that one of the scientists where I work checked out a bunch of gels using just a UV sterilization lamp one time when we thought our camera was broken. Since just eyeballing the gel like that works I'd be surprised if a regular camera couldn't at least get some kind of image.

[hanley]

Ethidium Bromide is fluorescent under UV light so Illuminating the samples with UV light and taking pictures using a standard camera in a dark environment may work

[wblack3]

Can add a bit more sterile 80% glycerol to the dye, as long as you adjust the dye buffer so it still ends up at 1x. Helps the samples sink to the bottom of the wells and stay there until it runs into the gel.

Also, less dye means you have less annoying 'dark bands' appearing on the photo.

Metacresol green and bromothymol blue spring to mind.

I think I used to add EB directly to the dye buffer, not the gel or tank buffer.

[wblack3]

Might do to give those combs a wipe with silicone oil or other coating solutions to make it more hydrophobic, stops the gel crawling up the combs, leading to wells that are seemingly deeper than they really are, leading to possible 'short circuit' running of the DNA over the top of the gel block.

W

[wblack3]

Good stuff!

Yeah, SYBRgreen is the way to go. You'll need different filters for the camera (~522 nm) and light source (~488 nm) with fairly narrow bandwidths for best results.

Oh, looks like you might be able to get strips of blue (488 nm) LEDs that run at 12 V. Sweet! These would be ideal as they would heat up less (you don't want hot gels!), don't need a filter, and don't 'fade' like UV LEDs do (surprisingly quickly!)

I like that there is less damage to the DNA from UV and ethidium bromide itself, especially when cutting out specific DNA bands for ligation into another plasmid. Also, UV is not so much fun to look at directly, even through nominally UV-blocking glasses!

Disposal of SYBRgreen is easier, too.

Have a look at TBE cf. TAE, it seems to last a bit better and run better.

There are other 'fast' running gel buffer systems.

Great that the molecular biology community is getting involved in Instructables.

Nice work!

Wes.

[ChrysN]

Great instructable on running gels! We use something called SYBR Safe(I think that is what it's called) as a DNA gel stain. It is not as nasty as EB.