Casting/pouring agarose gels - what to do and what goes on when you do so!
Casting/pouring agarose gels - what to do and what goes on when you do so!
longer blog form (refreshed from past posts): http://bit.ly/agaroses
more on agarose: YouTube: • Agarose science - composition, gelifi...
more on agarose gel electrophoresis: https://bit.ly/agarosegelcompare & http://bit.ly/agarosegelrunning & • Agarose gel electrophoresis - and a c...
We can separate biochemical molecules like proteins & nucleic acids (DNA & RNA) by size by sending them through gel matrixes. The gel’s like a 3-D mesh - bigger molecules travel more slowly because they have a harder time squeezing through mesh’s pores. So the molecules separate by size. Depending on what we want to separate (type of molecule & size) we make different types of gels. We often make AGAROSE GELS to look at DNA, & different types of polyacrylamide gel electrophoresis (PAGE) gels to look at proteins & RNA. We can play around w/amounts of the different components to get bigger or smaller pores depending on sizes of things we’re separating.
Unlike in PAGE, when we prepare AGAROSE gels, we are NOT doing the polymerization (chain-making) - that’s already done for us (thanks red algae!) Instead of adding strong, covalent bonds between individual subunits to form long chains called POLYMERS , we simply need to free existing polymer chains from their clumped-up-ness to form a more spread out matrix. The chains stay chains, we just need to change the ways they interact w/one another.
You can think of it like yarn. You have long strands of yarn that can be all balled up. That would not act as a good molecular sieve. BUT you can unspool the ball & knit a more mesh-like product that would. When preparing an agarose gel, its like you have lots of identical balls of yarn & you’re knitting them together.
1st you have to unspool them (& untangle strands that may be tangled up), which you can do by heating them up. This breaks the existing non-covalent interactions (the yarn unballs & untangles BUT you don’t “cut” any yarn). Then when you cool it down, new attractions can form. BUT, before they do, the yarn gets hydrated (puts on a water coat), so water gets trapped inside the mesh to give you a gel. Why? Let’s take a closer look at our “yarn”
Agarose’s “yarn” is a linear POLYSACCHARIDE (a type of carb) made up of repeating agarobiose subunits. agarobiose is a disaccharide (2 individual sugars (monosaccharides) linked together) made up of galactose (D-galactose to be precise) linked to a modified galactose monosaccharide, 3,6-anhydro-L-galactose connected 1-3, 1-4, where those numbers refer to positions on the sugars’ rings.
Each monosaccharide part uses 2 of its OH “legs” to link together (and some use others for modifications, etc.) but they still have “free ones” that love to stick to water. Water is “sticky” because of its ability to form hydrogen bonds (H-bonds). Covalent bonds form when atoms share pairs of electrons, but they don’t always share fairly. In water, for example, the electronegative (electron-hogging) O pulls negatively-charged electrons (e⁻) away from H’s so the H’s are partly ➕ & Os are partly ➖. Opposites attract, so H’s & Os of different molecules hang out in (individually weak, but collectively strong) non-covalent H-bonds.
Similarly, O’s in agarose’s -OH groups pull e⁻ away from H so O’s partly ➖ & H is partly ➕ and thus can form H-bonds w/water. So the “yarn” soaks up water, dissolving to form a “SOL” & when you “knit it” you undergo “GELATION” to get a watery mesh we call a GEL, an “infinite network” in which all the polymers are interconnected & water’s trapped inside.
Unlike actual knitting, knitting agarose mesh doesn’t take a lot of work. In fact, it undertakes molecular knitting as a form of relaxation. It’s the untangling that takes the energy, which is why you have to boil the agarose/liquid mixture.
Depending on the size of the DNA pieces you want to separate, you can make different percentage gels - the higher the percentage of agarose compared to water, the tighter the mesh, so the better separation you’ll get for smaller pieces.
If the percentage thing confuses you, you’re not alone - weight/volume percentage makes the bumbling biochemist moan! More on it here: http://bit.ly/weightvolume but for now just know that 1g/100mL = 1%. And, conveniently, we usually make 1% agarose gels (good for separating pieces of ~500-10,000 base pairs). So we weigh out 1g of agarose, pour it into 100mL of buffer (water + salts + pH-stabilizers) in a flask & bring it to a roiling boil in the microwave. I like to to microwave in 15-30s spurts & swirl in between to help w/DISPERSION (make sure all the agarose particles get a chance to meet buffer not just ones on outside of “clumps”)
In my lab we have a rubber oven-mitt-like thing so we don’t burn our fingers, which is much nicer (& safer) than the folded up paper towel I used for this purpose in undergrad!
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