The Principle of Pulsed Field Gel Electrophoresis (PFGE)

by: Biomedical and Biological Sciences

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hello everyone in this video I'm going to speak about pulses gel electrophoresis or PFD II I'm going to speak about the technique the principle of it and how does it work so let's first say what is PSG actually the sqe or pulses gel electrophoresis is a technique used to separate DNA or nucleic acid so it's very similar to a cure gel electrophoresis by the way if you don't know what it is gel electrophoresis you can go there is a previous video in my channel you can go there and watch a gern gel electrophoresis you can also find the link on the description of this video so pH EE is like agarose gel electrophoresis so it's an agarose gel it is a given and then as we fade in the agarose gel electrophoresis video we use elect an electrical field or an electrical current to separate did any samples or get again a fragment according to their size so we apply we here the battery we apply an electrical current with the negative negative charge on the top of the gel and the positive charge on the bottom of the gel and then because the DNA or the nucleic acids are negatively charged so they will move towards the positive charge and then the small molecules will all the small fragments or they obtain a will move farther and the larger fragments of DNA will stick in the gel earlier so what is now this is other gel electrophoresis Divini exact others an agarose gel electrophoresis we can separate the specific size DNA if the DNA segments are so large we cannot separate them because they will stick in the gel earlier Charlotte I will think about this idea in few seconds so first of all the PSG II is a technique we can say it's developed from I developed from agar gel electrophoresis because it depends on the same idea but the thing is that PF GE is used to separate larger fragments of DNA molecules you should know this so the main idea of PF GE is to separate large fragments of DNA now as I told you in a dress gel electrophoresis the small molecules or the small fragments of DNA migrate through the gel so they can migrate further and then the larger the fragment is the less it can meet migrate in the gel and then when the when the DNA fragments are so large they cannot migrate so they may form like a block like several fragments from a block on the top of the gel because they cannot be separated or they cannot migrate through the gel the thing is that a dress gel electrophoresis can separate up to 15 to 20 kilo base pairs again and again examples of up to 15 to 20 kilo base pairs but if you have larger DNA fragment and up to 10 mega base pairs so in 10 MB we can use a PFG now what's the idea of P a G or let's say what modifications did we do on agar gel electrophoresis in order to in order to make the poskim gel electrophoresis first of all if the concentration of the agar solution so as I told you before in the agarose gel electrophoresis video the concentration of the ideal solution is very important because it determines the pore size so you can imagine that the base gel is a matrix because agarose when it solidifies it forms a matrix and this matrix contains pores inside the more the solution is concentrated the smaller are the pores inside when the solution is less concentrated than the pores the pores will be larger so you can imagine that address electrophoresis is a bit like this is that it's a condensed matrix it's 2 percent matrix what we do in both still gel electrophoresis is that we are reduce the concentration of the address to 1% so it would be like like less convinced so the pore size inside the gel will be larger and so the large DNA samples can pass through these pores so this is the first modification second modification with it which is the most important is the direction of the electric field so the thing is that in agarose gel electrophoresis we apply the electrical current in one direction from negative to positive or let's say it's always from negative to positive but let's say from the top of the gel to just bottom of the gel in a vertical way what we do in pots will get an a conversion if that we change the direction of the electrical field to time the first one is in this direction and the second one is in that direction with 60 degrees angle between the direction and the vertical axis so we can say that the angle between the two other directions is 120 degrees angle now what why do we do this I'm going to tell me in a few minute but let's first so these are the two most these are the two important modifications in Pottsville gel electrophoresis mount lists let's see how do we do or what's the protocol of doing possible in gel electrophoresis throughout the performance first we should have the bacterial cells because we want to take the DNA samples from bacteria or from any kind of cell so we have a cell culture we take this cells from itself culture and we put the cells inside the agar solution so in this tube I have an I have water I add agora so I have an Agora solution I should dissolve the Agora and then I should have the this cells inside the Agora solution now what I have is I have either a solution with the cells inside so I put the solution in a special container to get like a something we called the plug so this is a plug it's a gel it's an agarose gel and it contains this film inside now I take these plugs and what I do is that say I apply some lights in the cell lysis to the plugs by applying or by treating the plugs with life solution but what's going to happen then is that these cells are going to be live so the cells are going to open and I'm going to get the DNA out of the cell so DNA extraction everything is happening inside the gel inside the plug now I will add the plugs with the DNA samples inside so I change the color because here I have plugs with cells inside and here we like the cells and get the DNA out of the soap now what what are we going to do now is as we did in agarose gel electrophoresis we don't need the DNA as it is we want to cut the DNA in smaller friend or in smaller signal because we are interested in one gene let's say or in one chromosome so now I want to cut the DNA to get the gene of my interest and to do this I apply restriction endonuclease enzymes as I told you in a restriction endonuclease infant or enzyme that comes again in specific places what are we going to get them is the the plot with the degraded DNA or the cut DNA it's not degraded it's cut to smaller pieces inside of course this be DNA are very large and because of this we are applying postive gel electrophoresis another question you might be asking is why did we do the DNA extraction and cutting inside the gel so why don't we just director as we did in agarose gel electrophoresis why didn't we apply a cell license and and DNA cutting in a normal solution and the answer for this is that high molecular weight DNA so what we are dealing with in Pottsville electrophoresis is high molecular weight DNA they are easily clipped through sharing and import in very high solution viscosity so they are easily pleased in high solution viscosity and because of this is easier to apply the cell lysis and the DNA cutting in a in a in a plug which is a very high which is a high viscosity solution now what we do is that we insert these plugs inside the width of the gel so normally in a gorilla electrophoresis what we did is that we apply the solution directly in the weld here we apply the plug so we insert the plug inside the well inside the well and then we apply the electrical current in not in one direction but in several directions so what's going to happen is that we are going to apply the electrical electrical current in this way so this is the first direction and this is the second direction and the third direction it's always from negative to positive because as you know the Na are negative and they migrate specifically from negative to positive the angle we are getting as I told you before is 120 degrees so it's a optic wearing array orientation and angle and the question now you might be asking why are we doing this why are we changing the direction of the electrical current so the principle of PF GE is that DNA the DNA segments elongate in the presence of an electrical filtering either gel electrophoresis when we apply the electrical field or the electrical current the DNA signals start to elongate and the thing is that the smaller gain a sigma a longer a elongate faster the relaxation rate depends on the site so the larger segment they need more time they need time to be elongated let's see it this way so this is a small DNA segment let's say it means that like several time and like a several second to be elongated but as much as the Sigma is when it's larger it means more time to be elongated and when I have very large DNA segments that like as I told you up to ten and B or major base pairs it means it needs a longer time to be elongated and then in the a question electrophoresis the time we apply the principle or the technique is not sufficient for the large DNA segments to be elongated and because of this they block at the top of the gel they don't have the time to elongate so what they are doing in PSG II is that we increasing the path of the samples inside the gel so how are you are we doing this in Afghanistan electrophoresis we apply one direction like this we apply one direction of the electrical field but in PhD II what we are doing is that we are taking the samples to the right and then we are taking them back to the left we are sending them to the right and then you are taking them back to the left so at each time what each time we are changing the electrical and the direction of the electrical field we are giving the larger than a fragment and more time to be elongated so imagine that they are elongated in one like in one straight line they don't have the time to be elongated but when they are taken in several directions they have more time to be elongated like this so it's like you can imagine the process is that we don't we are not moving them in one line but we are moving them like in a we call that we call this like in the zigzag path so we are increasing the start of the DNA samples inside the gel the thing is that we have separate parameters to to ensure that when we are applying a positive gel electrophoresis so first is the voltage which we which is measured by V over C and normally the voltage used is 60 V over C R U we should pay attention that the voltage applied in this direction and this time should be exactly the same voltage and time applied in this direction why because we need two DNA's to get back to their path so we so like I have this sample this type of should stay in this path so when we are taking this in the right we should ensure to take them back to the left so we should apply the same voltage at the same time so the thing is that the voltage as I told you we use 6 V over cm the transistor found that like a smaller voltage like 4 or 3 B over so or let's say 5 V over C M is better so larger than a fragment why because when we apply when I apply like a small voltage then let's say 3 V over C M then the samples will will move very slow because the voltage is is we are using a low voltage so the samples will will move very slow and when the samples move very slow they have more time to elongate smaller than larger fragments of DNA they have more time to in long gate so the smaller voltage used is the best for larger game if fragments but then we need much more time to do the process the second parameter we should take care about at the angle so normally the angle we are using is 120 as I told you but all the scientists found that the smaller angle used so like 160 is better for also for enlarges fragments yeah so when we use it as a smaller angle then it's better for larger fragments because also they will have a more like a more time to be elongated the third parameter is the switching time the switching time is the time from here to and from this point to this point so it's this time a plot it's the time applied in one direction there from this point to this point or from this point to this point this is what we call the switching time and exactly the same way or what I told you for the voltage the the longers of the switching time is the better for largest fragment because they will have more time to be elongated you should know something that when we when you apply the ideal parameters for larger fragment so when you apply a small voltage with a long switching time and small angle so this these parameters are as ideal for large DNA fragments then these then you are getting high resolution separating for large fragments but then these parameters are not ideal for small fragment so if you have in your sample large fragments and small fragments so you should find the ideal parameters for both of them the foreign-funded parameter we should take care about is the temperature of course because P fge is a very long process and sometimes it takes overnight so you should ensure that the temperature of the buffer is always in a certain range and sometimes we use pumping of the running gear buffer to the fluid Shearer or sometimes we stop the run and we change the buffer completely so these are the most important parameters for PF GE now we are getting the results like this and I wanted to show you this picture because the year we have results from the same sample so here are the same sample but different switching time so here we are using 45 seconds or as a switching time you can see that the smaller fragments are better separated but the larger fragments are not very good super is like a not perfectly separated when we increase the the switching time then now the largest fragments they have more time to be separated so you are getting a better resolution for let's say larger fragments and when we use 90 seconds of switching time then it's perfect for very large fragments why am small fragments are not very good suppurative so this is a been an example about how the parameters can can change see the result we are getting this is everything I wanted to tell you about bobstine gel electrophoresis I hope you enjoyed the video if you really enjoyed this please like and share if you have any questions you can write in the comments if you have any suggestions for other topics you can also write in the comments and don't forget to subscribe the channel



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This video provides the best explanation about Pulsed Field Electrophoresis, it helps you to fully understand the principle and the technique. Watch also Agarose Gel Electrophoresis: https://youtu.be/dSFQHt-zIQs

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