How to Load an Agarose Gel

you you after the jello sport it takes about 30 minutes at room temperature for the gel to cool and for the agarose matrix to solidify create something that we can add that we're ready to to load and use okay so we'll just put this to one side for now and through the magic of television I have a gel that I've already prepared so these gels are ready they're there they're solid and the way that we can test that so first if you're if you're being very cautious you can just blow on the gel and a liquid a gel that's still liquid you'll see that ripples form on the surface without disturbing the gel another thing you can do if you're feeling a bit more bold is you can actually just go ahead and touch that Jill to see it should it has you know about about the consistency of a piece of jell-o so I want to show you now before we load the gel I'm gonna I'm gonna sacrifice one of these gels just to give you an idea for how firm the agarose is and how careful we actually need to be while we are handling the agarose so I'll take it out of the mold here I'm gonna clear away this kind of this junk that sticks to the sides of the of the tray and here we here we have a finished mold so a few things you want to be careful everybody at some point in the lab has the experience of watching their gel slide out of the out of the mold out of the tray because there's just there's nothing holding it in on the sides so if the gel is wet it'll just it'll just slide right out and if you're walking around and paying attention it'll happen to you and your gel will break on the floor this is a 1% gel so as you can see it's it's reasonably solid right it doesn't break the first time that we touch it that's pretty solid exactly the consistency of your gel is gonna depend a lot on how much agarose is in it so this is a 1% gel I've seen 2% gels which are very very solid you can go down to as low as zero point eight percent which makes it very flimsy gel and lower than that the gel is probably too soft to use but the so the exact the exact composition of your of your of your gel is going to change how it feels so in general like unless you're dropping this thing on the floor it's it's it's it's pretty solid one thing you do have to be particularly careful of is where you put in the combs so where the comb goes the gel is particularly thin and so it's easy to crack the the bottom of the well where you want to load your DNA and if you do that you won't know until the DNA leaks at the bottom and your experiment is rode okay so that's what you want to be careful of so we'll we'll sacrifice this gel because I've been holding it so much I'm not sure if I can trust it and we will use his friend instead so I very carefully remove the comb take out the gel clear off the junk and then load it into the electrophoresis cell so this is the device that we use to actually apply the electrical current to the DNA molecules to pull them through the gel gel goes into place like that I take some more buffer so this is exactly the same buffer that I used to create the gel to fill the electrophoresis cell until the gel is covered now we're ready to conduct some electricity and then we've got to physically load our DNA samples into the wells that we created with the comb to to actually to just start the gel right so I've got some some DNA samples that I prepared earlier that we can use for that and I'm gonna use a little trick for preparing the samples to load which is I'll cut a piece of parafilm like this and lay it out on the bench parafilm side up so this is a place where I can mix very small volumes of liquid without having to use tubes right just because using tubes can be a bit of a pain you always lose a little bit of liquid on the sides of the tubes and also it's more difficult to see inside the tube so if I if I do the mixture like this you'll be able to see it to add a DNA sample to the gel the first thing that we need is what's called loading dye where's my loading dye here it is so this is a 6 X loading dye it contains glycerol glycerol is heavier than water and more viscous and that means when we mix our DNA samples with glycerol they will sink down to the bottom of the well rather than just floating away and and dissolving into the buffer that we prepared the loading guy also contains EDTA which we know stabilizes DNA and it also contains these dyes bromophenol blue and I forget the name of the other one that will migrate down the gel when we apply an electrical current and that all basically that allows us to estimate how long the gel has been running and and to see that the the electrical current is working right so a common misconception is to think that the the bromophenol blue dye stains the DNA directly but it doesn't it migrates independently of the DNA we're using the the cyber safe to stay in the DNA which you won't be able to see with your eyes we'll have to use fluorescence to see that later so the dye is supplied at 6x concentration which means that you need a minimum of one part of dye mixed with five parts of DNA but it actually doesn't matter if you use more than that and I generally do so here I'm I'm loading 5 microliters of dye just as these 3 little beads on my on my parafilm to that I'm adding 10 microliters of DNA sample and by pipetting up and down on the parafilm I can mix the DNA sample completely with the loading day up and down up and down up and down up and down up and down and one more that makes five microliters of dye ten microliters of DNA 15 microliters total this is ready to load on the gel which brings us to what is definitely the most difficult part of this protocol which is physically getting the DNA into these tiny little wells the truth is this actually takes some practice so unlike unlike most things in the lab this requires a tiny bit of dexterity to do correctly so I take my my 15 microliters of sample and so importantly I'm manipulating the pipette tip so that the sample comes all the way to the end of the tip right so I'm not leaving a drop at the end and I'm also not leaving an air gap at the end of the tip because if you leave an air gap that means you'll pipette some some air into the well and that will scatter your DNA you'll lose some if you do it that way okay gently load the DNA sample into the well so here it's important you want to put something of high contrast in the tray underneath the well so that you can actually see the well it's very easy for the welt itself to become invisible in the water and then beyond that I honestly I don't have many more tips it just takes a little bit of practice you will find that you're gonna ruin two or three agarose gels before you learn how to do it nicely and so plan your experiments accordingly but here we've got it three wells loaded the last thing we need is DNA ladder I'll add 5 microliters of letter to this gel DNA ladder is a collection of DNA fragments of standard known sizes that we can compare to our DNA fragments in order to estimate the size I'll add the ladder twice once on the left-hand side and once on the right close up the gel so this cover protects us from actually touching the electrophoresis cell while it's running because it's running at very high voltage and is therefore dangerous and I will plug this in and run it at a hundred volts for about half an hour

Channel: Synthetic Biology One

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Keywords: Synthetic, Biology

Id: u_KISppMWEQ

Length: 12min 9sec (729 seconds)

Published: Mon Mar 13 2017