Introduction: How to Prepare an Electrophoresis Argarose Gel

Picture of 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

Picture of 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.

Step 2: Prepare Stock Solutions

Picture of Prepare Stock Solutions

Preparing stock solutions allows you to caste multiple gels over a short period of time. This saves time and is more efficient.  Store the stock solutions in the gel electrophoresis area in your lab. Make sure to label each stock solution appropriately. If you already have these prepared in your lab go to the next step.

1) Prepare 1X TAE Stock Solution
a. Pour 20 ml of 50X TAE solution into 1 liter plastic bottle
b. Bring final volume to 1 liter with distilled water
c. Gently shake solution

NOTE : Many labs have 50X TAE solution on hand. If your lab doesn't, click on this link to find instructions on making one. 

2) Prepare 1X TAE + 1% Argarose
a. Add 250 ml of 1X TAE solution in 250 ml flask
b. Add 2.5 g of Agarose 
c. Boil in a microwave

WARNING: Solution will be very hot! Handle with care and use oven mitts.
 
After stock solutions are prepared, the next step is to cast the gel.

Step 3: Cast the Gel

Picture of Cast the Gel

The argarose gel acts as a medium for the molecules to pass through during electrophoresis. Wells, created by the comb, contain your samples during the electrophoresis process. At room temperature, the stock solution 1X TAE + 1% argarose gel is a solid. When casting the gel, the solution must be a liquid to form into the plate mold.

1) Place gel plate inside plate form
TIP:  Although the gel form supposedly protects against leaks, it is not full proof. When attempting to caste a gel for the first couple of times, try to tape the edges as shown in the second picture.

2) Turn front dial to tighten into place  

3) Attach comb to plate

4) Boil 1X TAE + 1% Argarose solution in microwave
This should take around 5 minutes

WARNING: Solution will be very hot! Handle with care and use oven mitts.

5) Pour hot solution into gel plate
For best results, make sure solution is completely liquid before pouring into plate. Supply enough solution to adequately create wells on the plate. 

6) Wait for solution to solidify
This should take around 5 to 10 minutes.

The mold should be solid before proceeding to load your samples. 

Step 4: Load Samples to Gel

Picture of Load Samples to Gel

Loading the gel can be tricky and time consuming depending on how many samples you have. When loading samples to the gel, find a way to stabilize your hand to avoid puncturing your gel. Your samples should contain approximately 2% dye.

1) Loosen dial on the plate form
Remember to remove the tape if you used any. 

2) Slowly and carefully remove the comb.
CAUTION: Avoid puncturing or damaging the gel.

3) Place plate with gel into gel box as shown.

4) Slowly pour 1X TAE stock solution into gel box

5) Add enough solution to fill the chambers and supply a thin layer of solution above the gel

If 1X TAE level does not adequately cover the wells, add more to gel box.

6) Pipette 10 µl of ladder into the leftmost well as shown in the third picture.
CAUTION: Do not puncture the bottom of the well with the pipette tip. For best results, insert the tip barely inside of the well.

7) Pipette 10 µl of samples and dye into the following well using the same technique 
TIP: Record the placement of each sample to the corresponding well. This will make things much less confusing when taking pictures of the gel.
 
The next step is to process the gel using electrophoresis. 

Step 5: Process Gel

Picture of Process Gel

Gel electrophoresis separates biological molecules based on size and weight by utilizing electricity. Polar molecules, such as DNA, move through the gel at different rates resulting in distinct bands. The longer the plate is exposed to electricity, the more distinct the bands become.

1) Using a plastic pipette, add several droplets of ethidium bromide to the front, middle, and back chambers of the gel box
WARNING: Ethidium Bromide is a known mutagen. Handle product with extreme care. Remove gloves after dealing with Ethidium Bromide and dispose of properly before proceeding to the next step.

2) Attach the lid to gel box.
Make sure to match up black electrodes with red electrodes.

3) Plug cords into power supply

4) Set desired voltage on monitor
This depends on your gel, but a safe voltage to use is 90V.

5) Push the run button and let electrophoresis run for 20-30 minutes 
TIP: You do not need to watch the gel during this entire process. However, wait until small bubbles form on each end before leaving the area. Periodically check to see if orange bands are moving down the gel from the wells as shown in the fourth picture.

6) Turn off power supply

The gel is now ready to be photographed by a UV Camera.
 

Step 6: Photograph Gel Using UV Camera

Picture of Photograph Gel Using UV Camera

Ethidium bromide is often used as in indicator because it binds with DNA. When exposed to ultraviolet light, it will fluoresce with an orange color showing the distinct bands of each sample. This can then be compared to the ladder. 

1) Unplug cords from power supply

2) Remove lid from gel box

3) Carefully remove plate and gel

TIP: To remove excess liquid between the plate and gel, use a paper towel.

4) Place gel and plate onto a UV camera

5) Take a picture of the gel using the UV camera

WARNING: Do not look directly into UV camera surface when on as it can be damaging to eyes.

After completing gel electrophoresis, taking a picture of the gel allows you to analyze the results of the procedure.The second picture illustrates an example of a photographed gel. The ladder is show on the left side. Distinct bands are illuminated in the picture. Wells are labeled to keep everything organized. Comparing the sample bands to the standard allows the user to estimate the size of each sample.  Electrophoresis allows you to analyze many different things, such as PCR  product or DNA size. Knowing how to perform this common lab procedure is key to being productive in a biological lab. 
 

Comments

wblack3 (author)2011-07-14

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.

DrRadium (author)wblack32015-11-14

TBE lasts longer than TAE and running buffer can be reused a couple times and is less prone to overheat. It may give sharper bands, but will start to smear when it gets depleted. Also, borate is less soluble than acetate so maximum stock is 20x not 50x and even then, precipitates tend to form over time. Borate can inhibit some enzymes, so when isolating nucleic acids for downstream reactions, I use TAE.

To minimize damage to isolated DNA, use long wave UV with fragnents being isolated for cloning. Better still use a system that doesn't use UV at all, or just clone fragnents direct from the cut or PCR and screen more. For multi-kB fragnents this is much more important. I was once unable to clone a 7kB fragnent after isolation regardless of visualization method. Went in the first try doing it blind without any fragnent separation.

Finally, for the most accurate resolution of fragnents by size, do not include EtBr in gel or buffer. Post stain after electrophoresis. It isn't common, but EtBr can cause aberrant migration in some DNA. Only seen it once, but it was a huge change in apparent size.

isacco (author)2011-07-25

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.

DrRadium (author)isacco2015-11-13

Running with the gel box in a fridge works well. Don't use a fridge where food is kept, especially if EtBr is in the running buffer or gel. Higher % agarose gels can take it hotter, lower % gels need it cooler, and low melt gels ( if anyone still uses it) should always be run cold

Lacustris (author)2015-04-27

Hi everyone!

I have a problem with my electroforesis pictures and I am so desperate about it because no one had helped me.

In attachment I am posting nice and wrong picture. Both are in same gene with same reagencies, I have a suspicion about the voltage.

My problem (as you can see) is that the genotypes are not clearly visible and I can make a lot of mistakes. I don´t understand why the bands are so fuzzy and not sharp.

Have anyone some tips or ideas?

Thanks a lot!

DrRadium (author)Lacustris2015-11-13

Also differences between the electrophoresis buffer and that in the gel can cause smearing. I like to make all the buffering use for both up in one batch. Usually it's ok to prepare them separately from high concentration stocks (the 50x TAE in this case), but be careful to measure accurately. A problem that should t be an issue but is, using 50x stocks prepared in different batches can cause smearing.

Not sure what standards were used, gel %, or lane IDs, so can't really troubleshoot these results much more than general principles. These look like test digests of plasmid preps but that's just an educated guess

DrRadium (author)Lacustris2015-11-13

Voltage can make a difference with small fragnents (under 1kb) when run for long times on low concentration gels. The band diffuses outward through the gel as well as migrating due to the electric field. Small fragments diffuse more easily and diffusion is more rapid in low concentrations of agarose.

geetawebspy (author)2015-01-27

Electrophoresis Dna Fingerprinting

http://www.bioolympics.com/

We are specialized in Gel Documentation System and we also focus on Electrophoresis of DNA and Electrophoresis DNA Fingerprinting.

DrMostafaI (author)2014-11-29

wonderfull

iudom (author)2013-03-25

noticeable equipment worth being sent to enhance the University of Nigeria Nsukka http://www.unn.edu.ng Laboratory

Kiteman (author)2011-07-12

Yay for hands-on science!

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

isacco (author)Kiteman2011-07-25

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 (author)Kiteman2011-07-14

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 (author)Kiteman2011-07-13

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 (author)Kiteman2011-07-12

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 (author)2011-07-14

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 (author)2011-07-14

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

ChrysN (author)2011-07-12

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.

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