These are some of the top questions surrounding gel electrophoresis and agarose gels. Learn about how to reuse/remelt your gels, how to calculate concentration, what volume gel to use for your specific tray, and more.
1.Can agarose be reused/remelted?
Agarose can always be melted back down for reuse. It’s a great cost saver too. However, like anything else, there’s a time when this is advantageous and a time when it’s not.
Any time you’re just running routine gels or doing a demonstration, reusing your agarose gel is a great option. But when it comes to the finer side of research: confirming findings, publishing, cloning, sequencing, extraction, Southern blot, etc., we recommend using a fresh gel.
Be aware that each time a gel is reused, background begins to increase.
Use caution when remelting low-melt agarose. Reusing low-melt agarose can influence concentrations since water is lost during the melting process.
Aside from remelting, some researchers run the bands off the gel and reuse the gel without remelting. Over time, however, the gel will lose conductivity.
2.How do you prepare agarose to be reused?
You can reuse your agarose gel a few different ways:
- Store in buffer and rerun
- Run off the gel
- Cast a gel with enough lanes to run a few different gels
For the first method (storing and rerunning), all you need to do is store your used gel in a container with TAE buffer. When you’re ready to run it, be sure to add in more ethidium bromide, run, photograph, and then store it again. Reusing a gel this way can make it last about a week, potentially longer depending on your preference.
Running bands off after use (second method) and then photographing works well. Again, remember to add your EtBr when doing this. And just know that for method one or two, you can only reuse these gels a few times. After a while, they will lose conductivity and have increased background.
For the third method, you can simply remelt your agarose and recast. However, be aware that there is some concern that microwaving agarose containing EtBr could release it in vapor form and later be inhaled. This concern is debatable among researchers, and remelting is still a common technique.
The last method involves using the extra unused lanes for additional runs. For example, let’s say your first run includes four lanes, and your second would include six, you can use a 16-well gel to run your first set, and then run the second in the remaining wells later that day. If you can’t get to it that day, wrap the gel in plastic wrap and store at 4°C.
3.Can GoldBio’s agarose be autoclaved?
Yes, agarose, including low-melt agarose can be autoclaved. This is particularly helpful when working with growth media that includes agarose. When autoclaving, use caution. It is a best practice not to autoclave material that contains ethidium bromide (EtBr) or products deemed mutagens that intercalate with DNA. Doing so might cause machine contamination.
4.How do you store an agarose gel that has already been ran/ agarose that contains DNA?
There are situations where you might need to hold off on photographing your gel. And often, it’s by accident that you learn that setting your gel in buffer overnight or even in the fridge will ruin everything. So what do you do?
The best solution we have found is to wrap your gel in plastic wrap without buffer and store at 4°C. Storing in buffer will likely cause your DNA to leach out into the buffer. Instead, this method should buy your analysis an extra day or two.
Another option, if you need to run your gel but can’t analyze it until the next day is to run it at a low voltage. The problem is that it only works when you’re planning for a delay. For unplanned situations and emergencies, it’s best to wrap and refrigerate your gel. This is also great when you are planning to later extract a band for cloning. In this case, just cut the band out, store it in a 1.5 ml plastic tube at 4°C. Remember to keep the tube properly sealed to prevent dehydration and do not use buffer.
5.Can you freeze Agarose gels?
Freezing your agarose gels causes icing and water buildup. Even if you’re trying to preserve a sliced gel for future extraction, it is best not to freeze it. Instead, wrap your agarose gel in clean plastic wrap without buffer and store at 4°C.
6.How long can agarose be stored?
A stock mix of agarose, in gel form without buffer, has a shelf life of a couple weeks. If no ethidium bromide is added into a stock mix and the mix is kept in good conditions, it might last longer than that.
7.What is the difference between agarose and polyacrylamide gels?
Application Differences: The answer has more to do with what you’re trying to accomplish. Agarose gels are great for larger DNA fragments and polyacrylamide gels are better suited for proteins and DNA fragments under 1000 base pairs.
You might be curious why agarose seems to be the common choice for nucleic acid analysis while polyacrylamide (SDS-PAGE) is used for proteins. You can still use SDS-PAGE for nucleic acids, and this is ideal when resolving very small fragments. Since agarose is better suited for larger molecules, proteins wouldn’t resolve as well with this technique.
Chemical and Structural Differences: In order to cast an agarose gel, the gel must be cooled to harden for use. Polyacrylamide gels, on the other hand, require a chemical reaction to set, and polyacrylamide cannot be reheated for reuse.
Pore size between the two gels also differs. A 1% agarose gel has a median pore size of roughly 100 nm. Keep in mind, this number is the median since agarose does not have a consistent pore size. The pore size for a polyacrylamide gel can range between 20-140 nm depending on makeup, and the pore size remains more consistent for polyacrylamide gels.Pour Differences: Agarose gels and polyacrylamide gels are poured and run differently. Agarose gels are poured horizontally while polyacrylamide is poured vertically.
Agarose gels are poured and run horizontally (pictured left) while polyacrylamide gels are poured and run vertically (pictured right).
Safety Differences: Because acrylamide is a neurotoxin there are safety concerns when pouring and using these types of gels. It’s important that people working with polyacrylamide gels wear proper protective gear and are trained.
Agarose gels are considered nontoxic. For this reason, they’re significantly easier to pour, store, use and analyze.
8.What is the history of gel electrophoresis?
Arne Tiselius has been highly credited for his electrophoresis work using a technique called “moving boundary electrophoresis,” which employed a U-shaped apparatus called the Tiselius Apparatus. During this early phase (around the 1930s and 1940s), Tiselius used electrophoresis to primarily study proteins.
This apparatus offered many advantages, and it had led to some exciting scientific breakthroughs. However, electrophoresis, at the time, still involved tedious work, a healthy supply of proteins and a rough optical system.
As time went on, the process became more and more refined. A form of zone electrophoresis, which used filter paper, became increasingly popular because of its ability to better separate proteins. Of course, this method also had disadvantages, mainly that proteins stuck to the paper.
Researchers began exploring different media to create a matrix for electrophoresis, including starch grain to develop a starch gel electrophoresis.
By the late 1960s, agarose was employed as a more common medium. With advancements in nucleic acid research and intercalating dyes, electrophoresis reached popularity by the 1970s.
9.Is GoldBio agarose safe?
While the acrylamide monomer in polyacrylamide gels is considered a neurotoxin, agarose gel is not hazardous. For full safety information about GoldBio’s agarose LE, refer to our safety data sheet (SDS).
Agarose gel is considered nonhazardous, but there are important first aid measures laid out on the SDS in case a researcher inhales, ingests, etc.
10.How do you determine DNA quality using agarose gel?
For genomic DNA, you can use gel electrophoresis to determine how intact your DNA is. Many researchers suggest running a low voltage (~75v) 0.8% - 1% gel for approximately 45 minutes. In this case, you’re looking to see if you have a nice singular tight band toward the top of your gel. This would be an indication of good genomic DNA. Smeared results are often an indication of DNA degradation.
Gel electrophoresis can also be used to identify contaminants such as RNA and contaminating genomic DNA. The key to evaluating your gel for contamination is the fundamentals of molecular weight separation: RNA, being a low molecular weight will run farther down the gel than your expected results. Genomic DNA, being a higher molecular weight, will not run as far as expected.
11.How do I calculate the percent of agarose to use for my gel?
Agarose gel concentration depends on the size of DNA you’re working with. This table shows what gel concentrations are appropriate for a given DNA size. In general, it takes a higher gel concentration to resolve smaller DNA fragments.
Your gel percentage is then determined by mass over volume. A 10% gel would be 10 grams of agarose in 100 ml of TAE. A 100% gel would be 100 grams of agarose in 100 ml of TAE. And a 1% gel would be 1 gram of agarose per 100 ml of TAE.
12.How do I determine what volume agarose gel to run?
Agarose gel volume is going to depend on the size of your casting tray. Small (mini gel) trays usually run a 40 ml gel. A gel tray approximately 9 x 11 cm would require a volume between 70-80 ml, and a larger tray measuring about 12 x 14 cm would need a gel volume between 120-130 ml.
13.How do you dissolve agarose?
Dissolving agarose for gel electrophoresis is a very simple process. Once you have determined the concentration gel you want to make, measure out the required mass of agarose powder.
Prepare a flask that can hold 2-4 times the volume of agarose you’re preparing.
Add the required volume of diluted buffer to your flask, followed by the weighed agarose.
Use a stir bar and magnet to mix the solution.
Once the solution is mixed, remove the stir bar. Place the flask in a lab microwave with a loose lid over the flask. You can also cover with plastic wrap and make a small hole at the top to vent.
Heat the flask in the microwave in bursts of 30 seconds. Swirl after each burst.
For a more detailed protocol, you can refer to this video or this protocol.
Keep in mind that you will need to choose the right agarose for your experiment. High resolution agarose is best suited for analyzing nucleic acid fragments less than 1 kilobases. General agarose LE is best for nucleic acids ranging between 50 bp – 25 kbp. And low melt agarose is ideal for separating larger DNA fragments (above 1000 bp).
14.What is pulsed-field gel electrophoresis (PFGE)?
In short, pulsed-field gel electrophoresis, or PFGE, is an approach for separating much larger nucleic acids that would otherwise not resolve using general electrophoresis. PFGE employs an alternating voltage gradient that moves fragments into a molecular weight-influenced pattern. The electrical currents switch between three different directions: one running through the central axis, and two on each side of the apparatus that runs 60 degrees.
In PFGE, the voltage is switched between three different directions: one running down the central axis, and two on each side that run 60 degrees.
The changes in electrical direction can change multiple times during the process and last for different designated times.
The basis of PFGE is still similar to the standard gel electrophoresis. At its core, both methods rely on molecular weight for separation analysis, and both rely on agarose gels and electrical currents to carry out the process. The differentiating factors are that PFGE does not rely on a consistently run voltage, the direction of current changes, and the time period of charge direction is regularly switched between three directions.
Applications for use include fingerprinting, epidemiological work and genotyping.
15.Should I use TAE or TBE?
If you’re not sure whether to use tris-acetate-EDTA (TAE) or tris-borate-EDTA (TBE), the table below might help you narrow down your decision. In summary, the choice depends on what you want to do, and the downstream processes you’re planning. If you still are uncertain, you can run both experimentally and see which one resolves better or best suits your specific needs.
Information gathered from BiteSize Bio’s article by Dr. Nick Oswald “Which is Best: TAE, TBE or Something Else?” Visit their article and many more for great information.
50x TAE Stock Solution Protocol
10x TBE Stock Solution Protocol
16.What does low EEO mean and why is that important?
EEO stands for electroendosmosis, and it’s a term that describes the electrically influenced movement of material through porous material. Low EEO allows increased mobility, reduced band distortion (caused by counter flow) and better resolution. It also allows shorter run times. Low EEO agarose or agarose LE enables larger particles such as viruses to migrate within the matrix.
17.How do I determine the voltage to use when running electrophoresis?
Voltage choice really depends on what your overall plans are. A higher voltage is going to cause faster migration. A lower voltage will cause slower migration. Another important note is that a higher voltage increases the temperature in the gel chamber significantly and can cause your gel to melt.
If you were in a situation where you needed to leave your gel for a few hours, running it at a lower voltage for a longer period is better.
Generally, gels should run between 75-150V for about 40-80 minutes. These settings will also depend on your apparatus and its accompanying instructions. Some machines may warn against voltage exceeding a certain point.
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