My First Taste of Lab Work: DNA Gel Electrophoresis
This key technique is a significant biotech tool—so I don’t think I did it justice by exploding a gel in the microwave.
While shadowing a biology lab at the University of Washington, the first thing I learned to do on my own was DNA gel electrophoresis (my first try failed spectacularly, but we’ll get to that later). It’s a simple technique with a simple goal: to separate DNA sequences by size. Though it may not seem so at first, this technique is applicable to many purposes and critical to many processes, making it one of the fundamental technologies used in biotech today.
Here, I’ll explore the following topics:
- An overview of the method of DNA gel electrophoresis
- A brief history of its development
- Its current applications and importance
- My personal lab experience and the process of gel electrophoresis
Let’s get to it!
Overview
In gel electrophoresis, molecules (in this case DNA) are moved by an electric current from one side of a clear agarose gel to the other. Because of the molecular structure of DNA, it is negatively charged and is attracted to the positive side. The gel is a dense web almost like an obstacle course, slowing down the DNA based on its size: shorter pieces easily slip through the gaps in the gel and advance further, while longer pieces are much slower.
Timing is important, too. If the electric current is shut off too soon, the molecules could be too closely packed to get accurate measurements of size; if the current stays on for too long, the DNA could run straight off the gel! This also means that length measurement markings can’t be printed on any stable surface — a slight time or voltage miscalculation can lead to inaccurate results. So, along with the DNA samples, a “ladder” (or “marker”) with preset DNA sequences of specific lengths is also run. That way, the samples can be easily and accurately measured by comparing their locations with the ladder DNA’s (see Figure 2).
You might be wondering about the black-and-white coloring of these results since, contradictorily, the gel is clear and so is the DNA. This colored effect is produced by a UV camera that detects certain fluorescent dyes like ethidium bromide or SYBR green. These dyes, unlike most, lock themselves into the DNA’s double-helix structure instead of simply floating around them in the solution and, as a consequence, moving independently of the DNA when the electric currents strike. So, in the UV camera, the fluorescent dye in the DNA produces a stark contrast against the background, making colorless DNA visible as white bands. Each of these bands is made up of billions of similar-sized DNA strands.
History
Now that the DNA samples are nicely sorted, they can be recorded, analyzed, and used for countless things in the world of molecular biology, a field that gel electrophoresis is and has always been intimately tied to. In fact, the concept of electrophoresis — the movement of charged particles in an electric field — dates back to as early as the 1930s, around the same time as the infancy of molecular biology. Over time, this technology developed: paper and starch gels were replaced by agarose gels in the 1960s, which saw competition from polyacrylamide gels a decade later; the 1980s brought about ladders and DNA fingerprinting, revolutionizing forensic science; now, with the advent of CRISPR and molecular cloning, gel electrophoresis finds new purpose in genetic engineering.
It’s impossible to say what will come next, but gel electrophoresis has grown and proven its use all throughout history, time and time again. It is critical to the development and exploration of molecular biology, so let’s examine some of its common applications!
Applications
1. Genotyping
A genotype is an organism’s complete set of genetic material. It is made up of a specific combination of alleles, or versions of each gene (for example, one allele can give someone blue eyes while the other gives them brown eyes). Each allele exists at the same spot in every person’s DNA and is mutually exclusive with each other. Because different alleles yield distinct outcomes, they vary in sequence and length. As a result, DNA gel electrophoresis can compare sequences from different organisms to determine which alleles are present based on length. This is extremely useful when the alleles of a certain gene don’t create visible or easily discernable traits.
2. Verification of DNA Constructs
Biotech is messy, and things often go wrong unexpectedly. PCR and molecular cloning are some common biotechnologies that play with cutting, extending, and attaching DNA segments. Often, they are also starting points for larger projects, so it’s crucial to confirm their success or identify the problem if the results are erroneous. With DNA gel electrophoresis, it’s easy to identify the parts of your puzzle through length and see if things came together smoothly.
For example, if a primer in a PCR failed to anneal, there would be little amplification, and you wouldn’t see any bands after gel electrophoresis — if there is one, check its location and hypothesize from there. If you’re trying to insert a gene into a plasmid through molecular cloning, gel electrophoresis can show you two shorter bands (they failed to attach), one longer band (success!), or something else (something weird’s going on…). These little lines can tell you a ton!
3. Forensic Analysis
DNA from a crime scene can be compared and matched to those of a suspect. People can also have DNA profiles, which are like genetic fingerprints. It’s true that the genotype of any human is more than 99% similar to those of another, but there are specific regions of DNA (short tandem repeats, abbreviated to SDRs) that vary highly from person to person in both sequence and length. DNA profiles, paired with gel electrophoresis, can identify individuals not only for forensic analysis but also for paternity testing, missing persons’ identification, genetic research, and more.
4. RNA and Protein Separation
The same concept of gel electroporation can be applied to RNA and proteins, too, and each comes with its own list of important applications. But, a few things must be tweaked, especially when it comes to protein gel electrophoresis (see Fig 4). First, proteins are complex, crumpled-up balls of amino acid messiness. So, there are an extra couple of steps and chemicals that must be added to unfold the proteins before they can even hope to make it through the gel. Second, fluorescent dyes like ethidium bromide don’t lock onto proteins like they do for DNA because proteins have a completely different structure. This leads to scientists thinking up a series of inventions that result in a vertical gel where the contents must be transferred onto a film, then covered with antibodies with fluorescent tags. There’s a lot more to it, though, so I’ll cover that in another blog.
Gel electrophoresis is a widely applicable and highly important tool for every biotechnologist to learn. So…
I tried to do it, and here’s how it went!
(Warning: dumb things were done)
Process — and personal experience!
The following image gives a nice overview of the process, so I’ll be following it loosely as we go (it’s not entirely accurate to what I did — I didn’t use masking tape and inserted the comb earlier — but it’s pretty much the same). Let’s dive in.
1. Prepare agarose gel
There are only three ingredients in a standard gel: TAE buffer, agarose, and ethidium bromide (EthBr).
TAE buffer is a solution made up of Tris base, acetic acid, and EDTA diluted in water — but most importantly, it was premade, so I didn’t have to worry about mixing those mysterious chemicals. TAE is used both in the gel itself and the buffer around it to maintain pH, conduct electricity, and prevent DNA degradation.
Agarose is a polysaccharide extracted from red algae that, once melted and then cooled, solidifies into a gel. In labs, it’s kept as a powder, and won’t dissolve until heated in near-boiling liquid (where it then starts foaming, making you worry if it’ll straight-up explode in the microwave). The gel’s density, the size of the channels DNA must pass through, and the thickness of the solution it's mixed into depends on the concentration of agarose.
So I guess it shouldn’t come as a surprise when, after I microwaved a TAE solution with 10 times the amount of agarose it was supposed to have, I found in the microwave a flask covered — both inside and out — with a sticky, hot, and smelly goo that must have boiled up and erupted out of the glass instead of a flask filled with calm, lightly simmering liquid. It then occurred to me that maybe the dot in front of the “4g” wasn’t a speck of dust but a period telling me I was supposed to add only .4g of agarose.
I was inclined to blame the handwriting for my mistake, but it was still me and not it who spent the next ten minutes cleaning the now-solid mess of agarose from the flask and microwave with my mentor. I was unspeakably abashed in front of the postdoc, but to my relief and gratitude, he simply helped me clean up with empathetic understanding.
The next time around, I made sure everything went smoothly. It’s a simple process, really: pour 40ml of TAE buffer into a flask, add 0.4g agarose, microwave for a minute or so (while periodically checking the agarose’s state of dissolution by taking out the flask with gloves and swishing the liquid around), let the solution cool until the glass doesn’t burn you, add 2 microliters (ul) of EthBr, mix, and we’re done!
After making the gel, I was wondering why, when viewed under UV light, the entire gel didn’t glow. After all, I’d mixed EthBr into the entire gel itself. It turns out that although EthBr is always fluorescent, its weak glow is practically invisible until it’s bound to DNA, where it will intensify nearly twenty-fold. I assume the reason EthBr isn’t mixed into the components of the DNA samples directly is that EthBr is sensitive to light, so it’s best to keep it in a tube wrapped with aluminum foil until the last moment (and loading it into the wells with DNA is more of a hassle, plus, the concentration may be hard to control at that point).
I should also add that ethidium bromide has long been believed to be harmful because it’s a mutagen (it locks in between the rungs of DNA, so you can imagine it’ll cause some trouble in your cells), but recent studies have shown that this ominous red liquid is actually pretty harmless in low concentrations (but gloves should still be worn). Even so, some prefer to mitigate this risk by using an alternative dye like SYBR green, even though it may be less effective.
2. Pour gel
It takes around 10–15 minutes for the gel to cool and solidify completely, so it was still a warm, malleable liquid after I added the EthBr.
As seen in Figure 7, I put a glass plate into a larger one that walled off all four sides to make a mold for the gel (the larger one will be removed so that the top and bottom are exposed for the electric current to pass through). Then, to make wells for the DNA samples, I put the purple comb inside where the gel will be. Combs come in many shapes and sizes, and the one I was using in the image below fits the small gel mold with six wells, each 1mm thick (you can compare it to the long white ones above). It’s important to pick a comb that makes at least one more well than the number of samples you have so that there’s space for a ladder.
With the comb in place, simply pour the solution into the mold and pop any bubbles that appear. Then, let it sit. You’ll know it’s ready when the gel transitions from transparency to a gray translucency (shown above) and when a slight wiggle of the comb is met with resistance.
3. Pour buffer solution
After pulling the comb out of the newly solidified gel, I moved the small glass plate with the gel on it to the glass container where electrophoresis will actually happen. The container has a raised middle section where the plate fits, and the two deeper ends have wires running across them that connect to the power source. It’s important to make sure that the gel is oriented correctly so that the DNA doesn’t run straight off the wrong side of the gel (I’ve heard this happens more than you’d think…). I was taught to remember the phrase “run to red” to remind myself that the wells should be opposite to the red anode so that the DNA will, as they say, run to red.
Here’s Figure 1 again for a quick visualization.
Now, we have a gel sitting between the anode and cathode, but nothing to connect them. Here’s where TAE saves the day. TAE buffer conducts electricity well, so it’s used to fill the glass container all the way until it just covers the top of the gel. That way, the electric current will run right through the buffer, gel, and DNA to the other side.
4. Load samples
Before we load the DNA for gel electrophoresis, we must make sure that there are enough DNA strands in each sample, since the brightness of the bands in the results is determined by the amount of DNA strands present. Freshly extracted samples have far too few copies of each DNA strand, but PCR (polymerase chain reaction) can multiply them and raise those numbers up into the billions in the short span of a few hours. Just what we need!
If you’d like to learn more about this remarkable technology, check out this blog on PCR!
After PCR, the samples are ready to be loaded. With a micropipette, I put 10ul of every sample into their corresponding wells, leaving the first well for 5ul of DNA ladder. It’s easy to accidentally puncture the gel with the micropipette tip while loading the samples, but I found that tilting the micropipette to the side helps to avoid that.
5. Run gel
Time for electrocu- I mean, *ahem*, electroporation!
I put on the glass lid of the container and connected the wires to the power source before turning the power on to 120V. Immediately, I see small bubbles rising from the wires and the dye starting to, very slowly, run to red. And by ‘dye’, I don’t mean ethidium bromide. There is a green dye — a mix of yellow and blue negatively charged dyes — mixed into the DNA samples as well. It’s the visible type and doesn’t fluoresce under UV light, so it acts as an indicator of where certain sizes of DNA would be at any point in time (the DNA itself is colorless). The yellow dye is much smaller — and therefore faster — than the blue dye, separating almost instantly when the current is turned on. The DNA samples I was running should be slower than the yellow but faster than the blue, so I knew when to cut the power so that the DNA would be nice and centered in the gel.
6. View results
After about 13 minutes, I cut the power to the wires, slipped the glass plate out of the container, and put it into the dark chamber in the UV camera machine thingy (didn’t get a picture of it, sorry). I texted my mentor to come deal with the photography technicalities, and a few minutes later, out came a picture!
Although the only thing that EthBr highlights is the DNA, EthBr is present throughout the entire gel, so interruption from the wells and the blue and yellow dyes show up as darker spots where the faint glow of EthBr not bound to DNA is muted. See Figure 7 for an annotated version of Figure 6.
Here’s what’s happening in Figure 7: the bright bands are DNA (ladder on the very left), the outlines of the wells can be vaguely seen at the top, and the large, darker bands are where the dyes are. These results show that PCR was successful in all but samples 1 and 4 (sample 9 was the negative control, so I didn’t expect anything to be there). The smaller, fainter bands below the bright ones are the PCR primers; meanwhile, the full, original DNA extracted directly from plants can’t be seen because their numbers are too few.
The results aren’t perfect, but my first DNA gel was a success!
DNA gel electrophoresis is a special one for me; not only is it a foundational and fundamental tool in molecular biology, but it’s also the first biotech technique I learned, messed up, retried, and succeeded at in a professional lab. It sure wasn’t the last one, though — I spectated, experienced, and attempted many new and wonderful things while shadowing at the lab, and I’ll share them all with you over the next couple of blogs!
