A note on electrophoresis in my lab
Hello everyone,
So as I wrap up the final PCRs for my current algal strains in preparation for my second round of DNA sequencing, I thought I'd go over how I perform electrophoresis, an essential step in between the synthesis of DNA by amplification and the sequencing of that DNA.
Electrophoresis is a technique used in the lab to visualize DNA (as well as RNA and proteins, but that's not relevant to my research at the moment), and it serves several purposes. First of all, it confirms that there is DNA present in a sample; in my case, this is to check the efficacy of my PCR reagents, but it can also be used for other purposes, e.g. whether or not DNA was successfully extracted from cells. Electrophoresis basically works by taking a small slab of gelatinous material and "filtering" DNA through it. This is typically done by passing an electric current through the solid gel; as DNA is negatively-charged (due to its phosphate backbone), it is attracted to the positive electrode and, if placed in a depression within the gel, will migrate through the pores of the gel towards the source of the positive charge. Depending on the material the gel is made out of and its concentration, the pore size and quantity can be changed, which will affect how the DNA travels through it. Generally, smaller pieces of DNA migrate faster, and larger pieces migrate slower, which explains a second important use for electrophoresis: determining the approximate size of DNA fragments present in a sample. By comparing a sample of DNA to a "DNA ladder", comprised of a mixture of DNA fragments of known sizes, it's possible to estimate how many base pairs are in the sample of DNA by looking at how far it moved through the gel relative to the ladder. The third purpose of electrophoresis is to determine the rough quantity of DNA present in a sample. When a gel is being solidified, it often has chemicals added to it which attach to DNA and make it fluoresce under a blacklight, UV, or blue light. The quantity of DNA in a sample is then directly proportional to how much the DNA "glows".
Lots to take in, but basically as a summary, electrophoresis helps make sure the right DNA is copied, and in a large enough quantity for sequencing to work.
In my lab, I use electrophoresis equipment loaned to me by a company called Accuris Instruments. Although they haven't paid me to promote their products, I can say that they have worked exceptionally well for me in the home lab setting, due to their compact size, intuitive and easy interface, and durability. This is what the equipment looks like:
The left two items constitute the MyGel Mini Electrophoresis System. The main unit, pictured center, has a power supply to provide current, a little platform on which the gel sits, and a tank surrounding it, which is filled with a solution to help carry the current through the gel and move the DNA. The far left has the gel casting system, which is built to mold a rectangular-shaped gel and hold a comb in it to form wells (more on why that's important below). On the right is the SmartDoc Imaging System, which is used to photograph the DNA in the gel. The gel is placed inside the base, which is then covered to prevent outside light from entering; blue LEDs inside illuminate the gel, which causes the DNA to fluoresce, and a smartphone is placed on the top to take pictures through the aperture of the cover, where an orange filter reduces blue light so that only the glow from the DNA is visible.
And here is what a completed gel looks like:
The gel is the faintly-illuminated green rectangle, and all of the brightly-colored bands inside it are DNA! The DNA bands are restricted in their width, and constitute what are known as lanes in the gel. This is because the dense sample is originally pipetted into a "well" (the row of holes at the top of the gel) and sinks into it, so when current is applied (positive electrode below the bottom of the gel), the DNA simply travels straight downward through the gel. The leftmost lane holds the DNA ladder, and it has multiple bands of DNA. Reading upwards, these represent DNA of fragments 100, 300, 500, 750, 1000, 1500, 2000, 3000, and 5000 base pairs long; there is more DNA of base pair lengths that are bolded, so the bands containing these DNA are brighter. Using this ladder as a metric, it is possible to see that the first few samples of DNA on the left line up almost directly with the 500 bp band, and are thus around 500 bp long; the next group of samples fall between 750 and 1000 bp in length. It's also notable that in the first group of samples, the leftmost two samples have more DNA than the right three, because their bands appear brighter.
This is another example of a completed gel, although it is not as pretty as the first one:
In this case, there are several bands which are very faint, indicating low quantities of DNA, and some reactions that barely produced any DNA at all.
So, that's a general overview of what electrophoresis is, how it works, and how I use it in the lab. Stay tuned for future updates!
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