An agarose gel is often used to separate DNA and RNA molecules primarily based on their dimension. It’s a generally used method in most biology labs, and whereas pouring and working an agarose gel ought to be a easy and routine process, there are a stunning variety of agarose gel errors you may make.
Listed below are 5 frequent culprits of an agarose gel electrophoresis gone incorrect and how one can keep away from these rookie errors!
High 5 Agarose Gel Errors
1. Utilizing Water As a substitute of Buffer for the Gel or Working Buffer
Agarose gels are solid and run utilizing TAE or TBE buffer. Since each buffers are clear liquids, it’s straightforward to mistake them for water. If water is used, the gel will soften shortly after making use of a cost to the gel field—say goodbye to these valuable DNA samples!
As one commenter mentions, you additionally don’t wish to unintentionally use buffer that’s too concentrated. So be sure you accurately dilute that 5X TAE earlier than including it!
Choosing the proper buffer can be essential. So be certain these bottles are clearly labeled, and you’ll simply keep away from this straightforward error in electrophoresis.
2. Forgetting to Add Ethidium Bromide (or One other DNA/RNA stain) to Your Agarose Gel
In a rush, it’s straightforward to neglect so as to add ethidium bromide to your agarose earlier than casting the gel. With out it, in fact, will probably be unattainable to visualise your DNA. Fortunately, you possibly can rescue an unstained gel after it’s executed working by soaking it in buffer containing ethidium bromide.
In reality, some folks even favor to stain it afterward, although you’ll then have a big quantity of ethidium bromide waste to eliminate (fortunately, it may be reused a couple of occasions earlier than you’ll have to do away with it).
Different stains are in fact out there. Try this helpful information.
3. Utilizing the Unsuitable Share (or Sort) of Agarose
The usual proportion of agarose used to separate DNA is 1.0%. Utilizing a better proportion of agarose in a gel enhances the decision of smaller bands. Compared, a decrease agarose proportion permits the smaller bands to run via the gel shortly, thus providing you with higher decision and separation of bigger bands.
If the incorrect proportion is used, it may be troublesome to visualise your bands. Watch out when utilizing a proportion of agarose under 0.8%, as these gels will change into weaker and far more liable to breakage.
Low melting level agarose can be utilized for specialised functions, equivalent to in-gel ligation.  These gels are very “soupy” and fragile, even at comparable percentages, so be sure you seize the appropriate reagent earlier than mixing up a batch of agarose.
4. Mixing the Leads Up from the Energy Supply
One of the irritating (however best!) errors is to unintentionally change the leads from the facility supply in order that the gel runs backward.
Because the wells are so near the tip of the gel, you’ll more than likely lose your whole samples in case you don’t discover the error instantly.
It’s not a foul behavior to hold round and verify your gel inside the first jiffy of beginning electrophoresis, which additionally offers you an opportunity to verify there’s a present flowing (simply verified by the bubbles coming off the electrodes).
So, in case you discover your bands heading within the incorrect course earlier than it’s too late, simply change the leads and run it again the opposite means.
5. Dropping your Agarose Gel on The Method to the Imager
Severely—don’t let this occur to you. Use an acceptable container to carry your gel, and don’t overload your self on the way in which to picture it. It’s higher to make a number of journeys if wanted.
Nevertheless, in case you do drop it, all shouldn’t be misplaced. you may be capable to rescue the gel relying on the appliance, the diploma of injury to your gel, and your puzzle expertise! Do bear in mind to scrub up the spill correctly although!
Agarose Gel Errors Summarized
That’s our high 5 agarose gel errors. Whereas we might have all made a few of these errors, luckily, most of us don’t let this stuff occur greater than as soon as.
What classes have you ever discovered out of your mishaps with agarose gels? Tell us within the feedback under.
- Upcroft JA. (1993) Comparability of properties of agarose for electrophoresis of DNA. J Chromatogr. 25;618(1-2):79-93
This text was initially printed on 25 March 2011. Reviewed and up to date June, 2022.