A lot of molecular biology involves working with pieces of DNA, such as those we might get from using PCR to copy specific sequences or using restriction endonuclease to cut DNA up. Agarose gel electrophoresis gives us a way to see what’s in there (for example, is there a band of the size you’d expect if a particular mutation was present) and, if we want to, use gel extraction to take them those pieces out & work with them. 

text from June, video partly new, partly from June – I added more practical and “what it really looks like” stuff, as well as more comparison to SDS-PAGE. more on SDS-PAGE here: https://bit.ly/sdspagepractical & https://youtu.be/FgXlPAKVSGY

After the video there’s a brief written version and some graphics and you can find much more detail here: http://bit.ly/agarosegelrunning 

The basic idea of this technique is that you separate pieces of DNA by size by using electricity to send them swimming through a gel mesh made out of the sugar agarose. DNA has a negatively-charged backbone, so it’s motivated to swim towards the positively-charged end of the gel, but it won’t have an easy journey! The mesh acts like a molecular sieve, slowing down bigger molecules more. So, when you turn off the electricity, the bigger pieces won’t have traveled as far and, when you visualize the DNA (typically by using a fluorescent DNA-binding stain and a UV light) the bigger pieces will show up as bands closer to the “top” of the gel and the smaller pieces will show up as bands closer to the “bottom” of the gel.⠀

note: “Top” and “bottom” can be a bit weird if you’re used to running SDS-PAGE gels. SDS-PAGE stands for for Sodium DodecylSulfate PolyAcrylamide Gel Electrophoresis, and it’s a related method we use to separate proteins by size (length of their amino acid chains). SDS-PAGE gels are thin and run vertically, sandwiched between two glass plates, so “top” and “bottom” make more sense. Agarose gels, on the other hand are thicker slabs that are run horizontally without the glass plates. Since you’re running horizontally, “top” just means “the end nearest the wells where you put in your samples” and “bottom” refers to “the end furthest from the wells.” In both cases, smaller things travel further and thus will be closer to the bottom. Assuming you set up the electrodes correctly!⠀

The whole “electro-“ part of electrophoresis relies on electricity, the movement of charged particles. When you set up an electrophoresis gel, you use a power box to create an electric gradient running through the gel, with the positive charge at the bottom of the gel and the negative charge at the top of the gel. Opposite charges repel, so negatively-charged things (like DNA) will move through the gel towards the positive end. The positive end has the red electrode, so you can use the mnemonic Run to Red! to remember how to set up the gel (be careful not to set the electrodes up backwards or your DNA will run the wrong way, out of the top of the gel instead of through it!⠀

DNA has a natural negative charge so we don’t even have to modify it to get it to run. This is unlike the case with protein gels, where, since proteins have different charges, you you have to coat your samples with the negatively-charged (anionic) detergent SDS in order to give them a uniform negative charge to motivate their swim. ⠀

the linear DNA travels snake-like through the gel, sometimes getting coiled around some of the agarose strands as it goes (like getting wrapped around the basketball hoop rim) ⠀

This snakelike, or REPTilan motion has a cool name – biased reptation & the longer the DNA fragment, the more tangled up around the gel matrix it will get → experiences more friction → gets slowed down more. ⠀

And the tighter the mesh (smaller the basketball hoop radius & thus more hoops in the same amount of space) the more opportunities for tangling and the more the friction. You can change the meshiness by changing the agarose concentration. I usually use a 1% agarose gel (1g agarose in 100mL TAE buffer → microwave to bring to boil – pour into casting well → let set). 1% is good for separating pieces that are 400-8,000 nucleotides long. ⠀

You can decrease the concentration to make bigger pores better for separating bigger pieces) or increase the concentration to get smaller pores better for separating smaller pieces⠀

You can’t see the DNA fragments until you dye them with fluorescent dye & look at them with UV light (more on this here: http://bit.ly/2U4na9r ) But when you do, they’ll appear as “bands” with bands towards the end (furthest away from the wells) corresponding to smaller, thus faster-migrating, DNA fragments and bigger fragments closer to the wells. The bands that you see while the gel’s running are just tracking dye that helps you know how far the gel’s run. In addition to the tracking dye, the loading buffer has glycerol in it which is heavier than water so it helps keep your samples from floating out. ⠀

Then, when we’re at the UV light part, we can compare the sizes of the bands to the sizes of a “ladder” containing DNA pieces of known sizes that we run alongside it – it’s important to note that these sizes are for LINEAR, double-stranded, DNA – if you’re DNA’s not linear it will travel differently

much more here: http://bit.ly/agarosegelrunning

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