 Dear students, today we are going to perform restriction length polymorphism experiment, commonly known as RFLP. Firstly, this experiment was discovered by Alec Jeffries in 1984. This RFLP is used for the analysis of unique patterns of DNA fragments which are genetically different between different organisms and animals. Due to these genetically differences, we can detect these animals and organisms differently. These differences which are patterns which are present in DNA are called VNTR or Variable Number of Tendon Repeats. The genetic polymorphisms are inherited from parents to the offspring and there are some differences between every each and organisms. Due to these differences, RFLP exploits these differences and differentiate between species and even different persons. First we will discuss about the principle of RFLP. There are some certain enzymes which are called restriction enzymes which cut the DNA molecules from different sites. These different sites are called as recognition sites. There are many restriction enzymes, most commonly used restriction enzymes and their recognition sites are there. There are some enzymes and these are their recognition sites. Eqr1 it can cut the DNA from C-T-T-A-A and the other example is Hint3 which is and it cuts from the T-T-C-G-A and the third example is B-A-M-H1. It cuts from the C-C-T-A-G. These are the recognition sites or recognition patterns where these enzymes recognize these sites and cut the DNA from these sites and this is the principle of RFLP. For this we require DNA, some restriction enzymes, some buffers and other things. For the DNA extraction you can watch the DNA extraction videos from the video portal and after the DNA extraction we will need some enzymes, their buffer, some water and reaction mixture. Now we will prepare the reaction mixture for the preparation of RFLP experiment. We need DNA restriction enzyme and restriction buffer and water for the restriction mixture. As you can see we need DNA, we require one microgram of DNA and normally we have 50 to 100 nanogram DNA per microliter and if our DNA is 50 nanogram per microliter then we will require 20 microliter for the DNA and there is 10x buffer which is provided with the restriction enzymes and we require only 5 microliter of 1x buffer. We will dilute the buffer and add the buffer as a 1x and then we require restriction enzymes, normally we require 10 units of restriction enzyme which is equal to 1 microliter. For the different restriction enzymes the restriction units and the volume of the restriction enzyme is used differently and we will have a total reaction mixture of 50 microliter and if we add 20 microliter of DNA, 5 microliter of 1x buffer and restriction, restriction enzyme 1 microliter and we will take final volume up to and we will add at water up to 50 microliter and the final reaction volume will be 50 microliter for us. And after that the preparation of mixture we will add, we will put it into for incubation. The incubation time and temperature is different for different enzymes, normally it varies from 1 hour to 24 hours, we are using EcoR1 and we will put it for 1 hour and the temperature is required at, there are specific temperature for specific enzymes at that temperature the enzymes react most fast and accurately and normally we put it at 37 centigrade for the reaction time. If the temperature is different it will be mentioned on the restriction enzyme. Now we will perform this reaction, we have different things with us, we have two types of DNA with us and this is our reaction buffer and this is our restriction enzyme. We will prepare it in app, in app and off tubes. Now we will prepare our reaction mixture. For the reaction mixture we need DNA, restriction enzyme, water and reaction buffer. Here is our DNA, we will take two samples, one sample and other sample and this is our buffer and this is our restriction enzyme. There will, you should take care of restriction enzyme, this is a fridge item, you just take it from the fridge and after that simultaneously put it back. Don't, don't vertex the enzymes and don't vertex the reaction mixture. We will put these reaction mixture in our PCR tubes. We have two types of DNA then we will require two different tubes for the reaction. These are our reaction tubes PCR tube, PCR tube 1 and PCR tube 2. One for the DNA sample 1 and second for the DNA number 2. Firstly we will add H2O water in our reaction tubes. So we need 20 microliters of DNA, 5 microliters of buffer and 1 microliters of enzyme. This is total 26 microliters and we will add 25 microliters of water and I am taking yellow pipette and I adjust it on 24 microliters. As you can see, as you can see this is 24. We will take 24 microliters of water in two tubes. After that we will take DNA buffer, 5 microliters put it on 5 microliters. This is our buffer. While transferring these buffer take care, do not mix these solutions. We will add our DNA, this is our DNA 1. 20 microliters of DNA 1 will be added. This is 20 or adding the DNA vertex and short spin it. We will vertex our DNA samples and short spin them. There is a one precaution don't ever vertex our enzymes because enzyme is a protein and then if you will give it a shock on vertex it may will not be very useful after that. Going to vertex these samples with short spin. While putting in the short spin put your tubes in opposite directions. Now we will take our DNA number 1 and we will put it in our tube number 1. Now we are going to put it in tube number 1. There is water and buffer in it and we will mix it slightly with the pipette. Mixing will be done with the pipetting. We will not mix it on the vertex and after that we will just short spin it. Now we will put our DNA number 2 in tube number 2. We have water, buffer and DNA in our tubes now. Now we will add restriction enzyme at the last stage. This is our restriction enzyme and only 1 microliters of restriction enzyme is required. I will set the pipette on 1 microlitre. Now you can see it is on 1 microlitre. I will add restriction enzyme. I will go to the bottom of the tube slightly mix it and then I will pour this into the tube. For the new tube I will take new tip. I will add 1 microlitre of restriction enzyme and put it in second tube. I will go into the bottom and will mix thoroughly with the pipetting. Now our reaction mixture is ready. We will not vertex it. We will mix it by simple tapping and we will short spin it because some reaction mixture may be along with the walls of the tubes. With the short spin these mixture which is along the walls will be settled down. Now our samples are ready to be placed at a 37 centigrade for 1 hour. For temperature setting you can use water bath, you can use thermal cycler or you can use any other device with the temperature in which you can set temperature at 37 centigrade. Normally these tubes are PCI tubes and we put it in the thermal cycler. Now I will start the thermal cycler and place my tubes in the thermal cycler. And we will put our restriction mixture which is restriction enzyme our DNA sample and buffer and water. The final volume is 50 microlitre. Machine is taking its time and we will put it in the machine at 37 centigrade for 1 hour. After that we will do gel electrophoresis to see our results from where this restriction enzyme has cut the DNA. There will be different recognition sites in different DNAs and if there is one restriction site the DNA is cut into 2 pieces. If there are 2 restriction sites the DNA will be cut into 3 pieces. If there are 4 restriction sites then it will be cut into 5 pieces. Now this is ready. You can go to the browse method or new method. You can make a new program for it but I have already designed a program for it. You can see with the name of RFLP. I will open my program. There is only one step in it. One step at 37 centigrade for 60 minutes. Now I will put my samples into thermal cycler. Just open it. There is a 96 well plate but we are going to put only 2 samples into the thermal cycler. You can put these samples into any cell. We will close our machine and then we will start the program. There is a run option. Our reaction volume is 53 microlitre and our program name is RFLP. And we will just start new run. The reaction has started now. It will take 60 minutes to complete the reaction and in these 60 minutes restriction enzyme will act on the DNA and will recognize the recognition sites and will cut it into different pieces depending upon the species or individual of that DNA. On the differences of these recognition sites present in the DNA we can recognize the DNA from which species or which animal or interspecies it is related to. Our reaction is completed after 60 minutes and our samples are stored automatically at 4 centigrade by the thermal cycler. Our reaction is completed here. Now we will take out our tubes and we will do gel electrophoresis. I will just stop the reaction. There is run report. I will take out twice samples. For the gel electrophoresis please see the gel electrophoresis practical. Our experiment for RFLP has been done now. You have seen the results and at the end we are just discussed some applications of RFLP and some disadvantages. RFLP is used in the diagnosis of different diseases like cystic fibrosis and it is used to compare different sources of DNAs. If we get a DNA from cat or animal and another sample of DNA we will take the DNA from for example from human. We can detect the difference between these two DNAs with the RFLP because in human there are different recognition sites and in cat or animal there are different number of recognition sites. So the enzyme will cut it into different number of fragments. Due to this difference we can detect the source of the DNA. The RFLP is used for genetic mapping because different enzymes cut different types of animal different types of DNA into different fragments. Depending upon the size of these different fragments we can prepare genetic maps for different animals and organisms. And at the end we are going to discuss disadvantages some of the disadvantages of the RFLP. The RFLP is now an obsolete technique because it takes too much time and there are many steps in the RFLP. But new techniques like PCR you can get results in few hours for these different techniques. So that was all from the RFLP. Thank you.