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Horizontal Gel Electrophoresis

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Horizontal Gel Electrophoresis
Hello everybody this is Doctor. Vishal Trivedi from department of biosciences and bioengineering IIT Guwahati. And with in the electrophoresis we were discussing about the basic principle of electrophoresis followed by we have also discussed about the vertical gel electrophoresis and how to perform the vertical gel electrophoresis. So in today’s lecture we aregoing to start discussing about the horizontal gel electrophoresis.(Refer Slide Time 01:31)
So in the horizontal gel electrophoresis the electrophoresis in this gel system is performed in acontinuous passion with the both electrodes and gel cassette are submerged within the buffer. What is mean by the continuous electrophoresis is that the current is not flowing through the gel.Which mean it is actually continuous for example when you are running the vertical gel electrophoresis or if you recall you have the 2 chambers.
One is the top chamber where you are keeping the buffer ok and which is the negative electrode. And then you have the lower chamber where you have the again the buffers ok and then you have the positively charge electrodes ok. And in between these 2 chambers you have the gel actually which is been sandwiched within the glass plates. So here you have the polyacrylamide gel and because and so the current cannot directly go from the negative to positive electrode.Whereas current has to go through with this gel and then the gel is actually connecting the 2electrodes chambers. Compared to that in the horizontal gel you have a chamber where you havethe negative electrodes and then you have the positive electrode and then a gel slab is beensubmerged with in the buffer. So the buffer is on the top of the gel, the buffer is on the bottom ofthe gel and that is why the current can freely move throughout this system.So the current can flow throughout this buffer solutions and the buffer solution is alsocontinuous. Whereas in this case the buffer 2 buffer chambers are connected through the gel.(Refer Slide Time 03:32)
So the electrophoresis chamber has 2 platinum electrodes placed on both the ends and areconnected to the external power supply from a power pack which supplies a direct current or theDC voltage. So you have a electrophoresis chamber where you have a negative electrode and thepositive electrode on both the side. And then these electrodes are connected to the power cod tothe power pack.
And this power pack can supply the DC voltage of the any amount. The tank is filled with therunning buffer and the gel casted is submerged inside the buffer. There are additional necessariesneeded for casting the agarose gel such as comb. Comb is required to prepare the differ wellsthen we required the spacer, then we required the gel caster. So, how to run the agarose gelelectrophoresis?(Refer Slide Time 04:30)
So before discussing about how to run the agarose gel electrophoresis let understand the reagentas well as the instrument what you are required. So for the buffer the purpose of each reagentused in horizontal gel electrophoresis is are as follows. So you require agarose, so agarose is apolymeric sugar which is used to prepare the horizontal gel for the DNA analysis.Then you required the ethidium bromide, so the ethidium bromide is a dye it is actually going touse to stain the DNA. So see the so ethidium bromide is required for the staining of the agarosegel to visualize the DNA. Then you require the sucrose, sucrose is required for the preparation ofthe loading dye for the horizontal gel. If you remember we were using the glycerol in the case ofthe SDS page.So you have a choice either you use the glycerol or to the sucrose the purpose is that you want toprovide a high density solution. So that the, whatever you load in to the gel will remain withinthe gel. Then you require the tris HCL, the tris HCL is the component of the running buffer andthen you will require the bromophenol blue, bromophenol blue is a tracking dye to monitor the
progress of the electrophoresis. Now once you prepare the buffer and the reagents then you canactually perform the horizontal gel agarose electrophoresis.((Refer Slide Time 06:04)
To performing the agarose gel electrophoresis the first you have to cast the gel compare to theacrylamide gel where you are actually going to add the cross linker and then you are going to addthe temed and APS to induce the polymerization reactions. Here you are actually not going to dothat for casting of the gel. What you are going to do is? You just going to take the agarose. Soagarose is the sugar.So, when you boil this and it is polymer actually when you boil this sugar it actually going toform a jelly like solution. Just like as you might have seen when you cook the rice in your homeand if you boil the rice it actually give you some amount of starch. So it is agarose is also similarkind of carbohydrates so where you if you boil the gel it actually forms a jelly like solution. Andthen if you pour like jelly like solution into a tray it actually contains the comb when the jellylike solution cool down then you are going to have a gel slab.So for the casting of the gel different step to cast the agarose gel for horizontal gelelectrophoresis are given in the figure. The agarose powder is dissolve in a buffer either the testTAE or TBE buffer. And then you heated to melt the agarose. Hot agarose is poured into the gelcassette and allowed it to set. A comb can be inserted into the hot agarose to cast the gel well forloading the samples.
In few cases we can add the ethidium bromide within the gel so that it stains the DNA while theelectrophoresis is going on. So what you are going to do is you are going to take the requiredamount of the agarose into a beaker and then you boil this you can use the burner or you can usethe microwave. And then once it get dissolve then at this step you can actually have a flexibilityof adding the EtBr.And then you pour it into the gel cassette and before it gets solidified you can also put the combso that it is actually going to give you the wells. And once the wells are prepared you can loadthe samples on to that particular wells and you can able to dissolve the gel DNA to visualize.(Refer Slide Time 08:28)
Now the gel cassette is placed in the electrophoresis tank sub submerged completely and theDNA are loaded into the well with the help of a pipetman and run with a constant voltage. Soonce the gel is prepared then you can just take out the gel from the gel cassette put it into theyour your running buffer putting into the electrophoresis chamber and then you connect.Then you load your DNA and connect it to the DC power with the help of the power bank youcan supply the constant voltage and then you can dissolve the DNA into the agarose. And theDNA runs from the negative to the positive end and the ethidium bromide actually runs presentin the gel stain the DNA. Observing the agarose gel in the UV chamber shows that, the DNAstained with the EtBr as the orange color fluorescence.
So what happen in this case is that when you have a negatively charge electrodes and then you apositive charge electrodes and then you have a place where you can actually load the DNA. Sowhat will happen is the DNA because the DNA is negatively charge it actually runs in thisdirection because it runs towards the positively sized. Whereas the EtBr is a positively chargedye so EtBr is actually runs in this direction.So while they are running in opposite directions the EtBr is stain the DNA and EtBr is stains theDNA within the major and minor group which means the EtBr actually intercalate within theDNA. So you know that the DNA has 2 strand and if these 2 strands are actually having the basison the sides. So within the basis the EtBr is getting intercalated and as a result it actually givesthe orange fluorescence when you visualize them under the UV.And what you see is when you keep the agarose gel into the UV chamber that you are going tosee the orange color bright DNA glowing within the agarose. So there is no de staining steprequired because the EtBr only gives the fluorescence when it interact with the when interactwith the DNA and intercalate to within the basis. That is why there is no de staining step isrequired because the EtBr which is not interacting with the DNA does not give the orangefluorescenceAnd what you can see is for example in this particular representative image what I am what weare showing is that the these are the plasmid DNA which has being resolved on to the agarose geland they are showing you the intense stain. What you see is this blurry signal from the gel isactually the RNA. So RNA is never being get resolved and never give you the compact bandbecause of that the RNA is going to give you the hissy appearances.So with this we have given you the complete theoretical information how to perform the agarosegel electrophoresis and how to run the gel electrophoresis. But the theoretical information is notenough that is why I would like to take you to the laboratory where we have prepared a smalldemo clip where we have shown how to boil the samples what are the precaution you shouldtake when you are boiling the agarose solution and then how to pour it and then how to preparethe gel?
How to prepare the wells? How to load the DNA? What are the precaution you should take whileyou are loading the DNA in to the chamber in to the wells? And ultimately; how to visualizethese gel into the UV chambers? What are the precaution you should take when you visualize theDNA into the UV chambers because as you know that the EtBr UV and all these are dangerousfor dangerous for the human being. EtBr is the is a carcinogenic molecules.So it actually causes the cancer. So that is why you have to always wear the gloves when you areperforming the agarose gel electrophoresis. So in this demo students have shown you the how toperform the gel electro for the horizontal gel electrophoresis, the agarose gel electrophoresis.(Video Starts: 13:05)(Refer Slide Time 13:05)
Today we are going to give you the demo of the agarose gel electrophoresis which we used todissolve the DNA. So in a typical agarose gel electrophoresis separators what you need is youhave horizontal you have the buffer chamber where you can have the 2 electrodes. You can havethe anode which is the black color electrode and then you can have the cathode.This cathode and anode will go and house into the buffer chamber. Connecting these cords to thepower cords you have the 2 different cords one is the red cord and one is the black cords. Andapart from these you also need a gel caster where you can be able to cast the gel. So this is thesmall gel tray which you can use to cause the agarose gels. And apart from that you also have thecomb which actually can be used to prepare the well.
And besides this reagent what you need to perform the agarose gel electrophoresis you need theagarose powder which you can buy from the sigma or any other company of very high quality.Then you require the EtBr which is the staining dye and as you can see that the EtBr is beingkept in a vial which is covered with the foil because EtBr is light sensitive. And so it should beprotected from the light.And apart from that you also require the running buffer which is the 50 STA and so let us startthe casting of the agarose gel and performing the gel electrophoresis. Before you start thepreparation of the agarose gel what you have to do is you have to set the tray so that you can ableto pour the agarose in to that particular gel in particular tray and then you could cast.What you have to do is you have to take this tray you have to very carefully clean the trays andwith the water as well as, so that it will be free of any kind of contaminations and any kind ofbecause you know that the DNA is very sensitive for the DNA’s for the other kind of enzymes.Then what you have to do is you have to keep this tray in this way into this gel caster and withthe help of this screws you have to screw the gel tray.And you have to tight it and as you can see that I am tightening it with a both the screwssimultaneously so that it will be completely sealed. And then now once this tray got sealed andyou are ready to pour the agarose what you have to do is you have to first put the comb. Andwhen you put the comb what you have to do is you have to ensure that there is a enough gapbetween the comb as well as the lower end of the comb as well as the tray that space the agaroseis going to fill and that is how it is actually going to help you to prepare the well.So for the typical gel agarose gel electrophoresis what you have to do is you have to first preparethe 1X TAE buffer for a typical kind of small chamber. You required somewhere around 300 to400 ml buffer and for the gel also you require at least 30 to 40 ml. But so for a safer side what wecan do is we can just prepare the 50 ml agarose gel and for a DNA of around 1 KB to around 1KB you can actually run gel of 0.8% because as the DNA size will lower down you have to keepincreasing the size of the agarose percentage.
So for example if you are instructed to explore the you know composes or direct segmentation inthose cases the gel band or the DNA band what you are expecting is from 200 base weight tohigher molecular weight. So in those cases we normally run the agarose of 2%. But in the mostof the cases what we do is we run around 0.8% agarose gel and that is good enough to resolvemost of the DNA sizes.So for preparing a 50 ml 0.8% agarose what you need is you have to just wear the 0.4 grams ofthe agarose and then you have to boil it into the microwave and then you can prepare. So let usunderstand how to do that. Now what we have done we have weight the required amount ofagarose and then we have prepared the 1X TI buffer and some of the buffer we have poured intothe chamber itself and the for preparing the agarose gel what we have done is we have takenalmost 70 ml of the buffer because when you boil there will be always a loss of some buffers.So what we have calculated? We have calculated of 50 ml and now we are keeping 10 to 15 mlof more water because when you boil this you are going to buffer it the water is going to buffer itand that is why you are going to loose some water. So once you are you going to pour theagarose in to the buffer and then you are going to do the boiling. So what you can do is you canuse a microwave and you just turn on the microwave and what you have to ensure in themicrowave is actually boiling the agarose and melting it.So in this process what will happen is that the water will going to warm up and the agarose isgoing to melt. And once the agarose is going to melt it is actually going to swell and it is going totake up the water. And in that process it is actually going to make a viscous material or theviscous jelly like material and that is what is in the agarose gel. So but before because the it isgoing to boil it is actually can also come out from the from the beaker. So you have to keepopening and keep checking that is something that is not happening.And in between you have to always mix because the agarose is the made up of sugar. So it alsocan get charred if it get localized heating. And once your agarose is going to heat up then youcan be able to use that and you have to ensure that you boil it very cuddly. So that all thegranules what is present in the you know the agarose should be get melted. Let us check whetherthose agarose got melted or not.
For checking what we do is we have different kinds of the gloves which the gloves like is madeup of rubber and these are the gloves what you can use for touching the hot solutions because theour normal gloves what I am wearing right now is not good enough. So we have to heat up littlemore because still I can see some of the agarose granules. So once this step is over then you haveto just take out the agarose what you have to see that is the water is boiling right now ok andvapor are coming out.So this is actually rough estimate that you are going to loose somewhere around 10 to 15 ml ofwater in the process of boiling. Now what you have to do is you have to just let it get cooled forsometime and then you have to add the dye. So in this case we are adding the EtBr so once it getscool down you can just add the 3 microliter of EtBr and remember EtBr is a cast agent dye.So you have to very careful you always have to wear the gloves and you have to discard these tipvery thoroughly or very because you do not want to contaminate the environment. So this tip aswell as all the phosphor has to be discarded in a in a way so that it is not to out. So you add theEtBr and then you keep this tip in a secured space so that you will be able to throw this in a trashbag which is meant for collecting the carpentering material.Now your solution is ready and I can pour this in to the your tray. So you can just pour this intothe tray and we have to weight for sometime so that it should get solidify and then you can loadthe DNA and we can run the agarose. Now you can see that the agarose gel is being solidifiedand you can see that it is clear. So there is no granules or the granulate or the precipitate materialpresent. And now what we have to do is we have to remove this form and we have to keep in tothe chamber and then we it is going to be ready to use.So before you do that you have to add some amount of buffer into this tray. So what you have todo is you have to add some amount of buffer ok. And then you have to very carefully withoutmaking any movement in the longitutional direction you have to vertically uplift the comb intothe top chamber like this ok. And that actually is going to prepare the well. So what you can seenow is the wells in this particular agarose block ok.
And once you sure that there is no leakage and there is all these wells are good enough and thenwhat you can do is? You can unscrew this tray or the cassette and we are going to remove thecomb. So what we are going to do is? We are going to remove this into this chamber and then weare going to remove the comb very carefully you remove the comb ok. And then you are going tofill the chamber with the buffer.So when you fill it with a full buffer the gel is going to be submerged and that is why thisparticular type of gel electrophoresis is called as the continuous gel electrophoresis. Now whatyou can see is that we can see the wells in the gel. So if you see the gel you can see the wellactually and now we are going to load the sample into those well and then we are going toconnect the chamber to the electrophoresis power supply.And then it is going to perform the electrophoresis. To prepare the DNA sample what we havedone is we have to add actually 10X loading dye. So accordingly you have just take the DNAyou add this small amount of the buffer and then you add the loading buffer in such a way so thatit going to be 1X and then you are going to load the sample. So in this case we are loading the 25micro liter of the sample.So you are going to take the sample into the pipette ok make sure that there is no air bubble. Andthen you are going to visualize the gel and what you can see actually or sometime if there is aproblem in looking at a wells or there is a problem of visibility of the wells because sometimethe lower chamber or the lower background is also going to be transparent. In that case you canjust put a black paper at that at that place where you have the wells.And that actually is going to allow you to see the well properly. So let see how to load this soyou hold your pipette ok and then you bring your tip next to the well ok and then you load thesample. And what you will see is that the DNA is getting built into the well. So now see herehow I am loading the sample ok. So I have taken the 20 microliter and first I will do is I will takemy tip into the well and then I will load what you will see is when I am loading I will verycarefully slowly I am keeping my tip out ok.So that the whole well is going to fill with that tip but what you have to remember is that whileyour tip is or while your tip is inside the well you should not leave your plunger. So that it should
not create any air bubble because it that happen the DNA will come out ok. For now what wewill do is we will connect this to the cord so the black will go with the black and red will go withthe red. So before doing so we have to put the lid so that there will be no evaporation of thebuffer.So you just connect the black to black and red to red and now we have to turn on the power bank.So in a power bank you have a switch here which actually allows the turning on. Connected thechamber or the electrophoresis chamber with a power supply unit we are set it at 80 volt and nowit is running. And what you will see is once the DNA (()) (26:39) dye will reach to the end of thegel. Then we are actually going to remove the gel from the apparatus.And then, since we have only added the EtBr into the gel. The EtBr is going to run from thenegative side to positive side. Whereas the DNA is going to run from the towards the positiveside because the DNA is negatively charged and the EtBr is the positively charged. So they willrun in a opposite side while they are running the EtBr is going to intercalate within the basis ofthe DNA. And that is how it is actually going to stay in the gel at the DNA into the present in thegel.So that after that what you have to do is you have to take out this tray and put it into the gel docmachine and that actually will going to visualize. Then close the thing application nucleic acidethidium bromide exposure, optimal exposure or we can select manual also then we will acquirethe images. Now you can find here this is the DNA ladder this is the PCR amplified product.(Video Ends: 28:29)So in the demo which I have explained every aspects related to the agarose gel electrophoresishow to perform the gel electrophoresis? And with this I would like to conclude our lecture hereand in a subsequent lecture we are also going to discuss different variants of the electrophoresisgel electrophoresis and how you can be able to utilize them for exploring the answer or forsolving the experimental questions thank you.