 Dear students, today we are going to perform plasmid DNA isolation from bacterial cell culture. DNA, genomic DNA is a different type of DNA and it has a large size while the plasmid DNA is in very small size. So there is a different technique in which we use to isolate the plasmid DNA. In the plasmid DNA, we have three steps in which we isolate the plasmid DNA. While in the genomic DNA isolation, we most of the time have straightforward protocols in which cell wall or cell membrane is disrupted and nuclear membrane is disrupted and we get the DNA. But while getting the plasmid DNA, we get the plasmid DNA along with the bacterial DNA. So we have to avoid the bacterial genomic DNA and separate it from the plasmid DNA. There are mainly three steps in which we isolate the plasmid DNA from bacterial cell culture. In the first step, we cultivate the bacterial cells and we culture them for 24 hours at 37 centigrade. After that, we get the bacterial cell culture and we centrifuge it and we get the bacterial cells. These cultivated bacterial cells are then treated with different solutions to license the cell wall of these bacterial cells. We use isotonic solution like HCL, EDTA and we use the alkaline solution like SDS. In these, and we also add glucose in these solutions. These disrupts the cell wall and also denatures the DNA. The DNA of the bacteria and also the plasmid DNA is denatured. And in the next step, re-natured of the plasmid DNA and is occurred. We re-nature the plasmid DNA with the potassium acetate. Genomic DNA has a large size so it is not easily re-natured while the plasmid DNA is in a very small size and it is re-natured very easily. After the re-naturedation of this plasmid DNA, we get this plasmid DNA and we purify it with the alcohol and then we concentrate it to further use. In LAP we use a master kit for the purification of plasmid DNA. This gene jet mini prep kit is used for the plasmid isolation. It has a certain protocol and we will follow the protocol and isolate the plasmid DNA from this kit. This is the bacterial cell culture of E. coli which we prepared yesterday. We cultured it yesterday and it is now ready for the use and we will extract our plasmid DNA from this bacterial cell culture. These are E. coli cells. For the culturing of E. coli in LB media, you can see our another video. Now we will take 1.5 ml of the cell culture and we will centrifuge it. The bacterial cell will be pelleted down and we will discard the supernatants which will be the LB media. And then we will use these cells for the further plasmid DNA isolation. Now I am taking these two append of 1.5 ml tubes. I label these tubes as 1 and 2. Now I will shift 1.5 ml of bacterial culture into two append of tubes and we will centrifuge it at 10000 rpm for two minutes. I just open these tubes and I take the blue pipette. I will set it at 1000. I set it at 1000 microlitre. First I will dispense 1000 microlitre. And after that I will dispense 500 microlitre into each tube. This is cell culture. We will just open it. I have opened both the tubes. Just mix the cell culture before taking the culture because sometimes bacteria are at the bottom. This cell culture was grown with proper antibiotics so that we can confirm that the culture is properly grown. For culturing these E. coli you will see our separate video in which you will see how you will culture these bacterial cells. Now I will adjust my pipette at 500 microlitre and add more 500 microlitre in both the tubes. You can see it's 500 microlitre now and I will add more 500 into each tube. Before taking mix the culture. Discard the use tip. Cover the test tube properly and put it in this stand. Now I have taken this 1.5 ml cell culture into two append of tubes which was previously labelled. Now I will centrifuge these tubes at 10000 rpm for two minutes. The bacterial cell will be pelleted down while the extra lb media will be supernatant. I will discard the lb media which is supernatant and will take the bacterial cells. And from these cells I will extract the plasmid DNA. Now I will put these tubes into the centrifuge machine for two minutes at 10000 rpm. You can see I have set this centrifuge machine at 10000 rpm for two minutes. I will open it and put these tubes into the centrifuge machine. While keeping the tubes into the centrifuge machine make sure that you have properly balanced it. And properly close the centrifuge tube. Now the two minutes have completed and our centrifugation has been done. So we will open it and we will take our samples. You can see the bacterial cells are pelleted down. We will discard the supernatant which is most of the time lb media. And we take the pellet which is bacterial cells and we will proceed with this pellet. Now I am going to discard the supernatant and I will take the pellet. While discarding the supernatant make sure that pellet is not being discarded with the supernatant. You can see it is the pellet and now I will discard the supernatant from the other tube. I am making this with two tubes because you must have some balance while centrifugation in the centrifuge machine. In this tube you can see the pellet is more clear. These are the bacterial cells from which we are going to extract our plasmid DNA. Now our bacterial cell culture is ready for the further use. So we will proceed to our plasmid extraction kit which is thermo-scientific gene jet plasmid mini prep kit. These are the following solutions which are present in this kit. This is lysis solution, this is resuspension solution, this is neutralization solution and this is wash solution. So these solutions will be used in different steps. In first step we will add 200 microlitre of resuspension solution. I will take the pipette and we will set it at 200 microlitre. I will add 200 microlitre of resuspension buffer into our tubes. This is resuspension buffer. I have set my pipette at 200 microlitre. Now I will dispense 200 microlitre of resuspension buffer into our tubes. I am adding it to the first tube and now into the second tube. Discard the tip, close the cap immediately. Now I will close my tubes. These bacterial cells will be resuspended in the resuspension solution. We will mix it while vertexing it for at least 30 to 40 seconds. Mix it thoroughly so that all the bacterial cells are resuspended in the resuspension buffer. Now you can see these bacterial cells are resuspended. Now I will do it with the next tube. Now you can see these bacterial cells are resuspended in the resuspension buffer. After that I will add 250 microlitre of lysis buffer. You can see this is the lysis solution. This lysis solution will do the lysis of bacterial cells and bacterial cell wall will be disrupted. And all the plasma DNA and genomic DNA from bacteria will be out in the solution. So I have set the pipette at 250 microlitre. I will add lysis solution into both of my tubes. This lysis solution will break down the cell wall of the bacteria and all the plasma DNA and the genomic DNA will be available in the solution. I will just mix these tubes by inverting these tubes from 10 to 20 times. After mixing the lysis solution, now I will add neutralization buffer 350 microlitre in each tube. This is our neutralization solution from the kit. Now I will add 350 microlitre in each tube. Take the new tip, open the bottle and dispense one by one. Make sure that there is no bubbles while dispensing this solution. Don't touch the tubes, close the bottle and now close the tubes and mix it. At this stage there is genomic DNA of the bacteria present in this tube while the plasma DNA is also present. Now we are re-natured the plasma DNA which is in small size and easily re-natured while the genomic DNA of bacteria which is in large size is not easily re-natured. So we will pallet down the bacterial DNA and we will collect the plasma DNA from the supernatant. Now I will centrifuge this at 10000 rpm for 10 minutes and the chromosomal DNA will be palleted down and I will take the supernatant in the new tubes because our plasmid DNA is in the supernatant. I am putting these tubes into the centrifuge for 10 minutes at 10000 rpm. Open the centrifuge, put your samples in there. Make sure that the centrifuge is properly balanced, close the lid carefully, close the centrifuge machine and start. Now our centrifuge run has been completed and we will take out our tubes. Open the centrifuge machine carefully. You can see all the genomic DNA and the plasmid DNA is in the supernatant. We will take the supernatant and purify it while passing through at the column. We will take two separate columns, these are the columns with the centrifuge tubes. We will label our columns according to our tubes. As our tubes are 1 and 2 and we will label our columns as 1 and 2. We will take carefully our supernatant and pass through the column from tube number 1 to column number 1. While taking the supernatant, please don't touch the pellet. Now I am transferring the supernatant into the column. While going into the column, don't touch the column membrane. Now discard the tip and transfer the second sample into the second column. Add the new tip, open the tube and carefully took the supernatant. You can take around 600 and 700 microlitre supernatant or you can take even 500 microlitre supernatant. But take carefully and don't disturb the pellet. Now you can see the column is filled with the supernatant. Now we will centrifuge these columns and this supernatant will be passed through the membrane to the bottom. We will centrifuge it at 7000 rpm for 1 minute. Carefully place these columns with tubes into the centrifuge machine. Close the lid and adjust the centrifuge speed at 7000 and adjust the time for 1 minute. Close the centrifuge machine and start. Now you can see our run is complete. Open the centrifuge, take out the tubes with the columns. You can see our supernatant is passed through the column and this will be discarded. While our plasmid DNA is in the membrane of the column. Carefully take out of the column, take out the column carefully and discard this solution. Now we will put 400 microlitre washing buffer into the both of the tubes through the column. This is our washing buffer washing solution and I will adjust my pipette at 400 microlitre and will put 400 microlitre washing solution into the both the columns. While adding the washing buffer please take care that don't touch into the columns specially the membrane. Because it can puncture the membrane and your plasmid DNA will be lost. This washing buffer will wash our plasmid DNA which is stuck into the membrane of the column. Close the tubes carefully and again we will centrifuge it for 7000 rpm for 1 minute. Carefully balance the centrifuge machine, close the lid. As you can see it is already set at 7000 rpm for 1 minute and start. Now our run is completed again. Open the centrifuge machine, take out the columns with tubes. You can see the washing buffer has passed through the column membrane. While passing through the column membrane it has washed our plasmid DNA. Take out the column carefully and discard this buffer. Again take out the column carefully and discard this washed buffer. We will repeat this step again and will wash this plasmid DNA with the washing buffer 400 microlitre and will centrifuge it for 1 minute at 7000 rpm again. We will take washing buffer again 400 microlitre, open the tubes. The pipette is already set at 400 microlitre. Take the new tip and add the buffer into the columns. Don't touch the column and especially the column membrane while adding the washing buffer. Close the column caps and tubes and again centrifuge it for 1 minute at 7000 rpm. I have put the tubes into the centrifuge machine, close the lid and start the centrifuge machine. Again our centrifuge run is complete and we are taking out our tubes. You can see the wash buffer at the bottom of the tubes while our plasmid DNA is in the column. We will again discard this buffer, carefully take out the column and discard this solution. For the second tube again take out carefully and discard this solution. So now our plasmid DNA is in the column stuck with this column membrane. But there may be some extra washing buffer in this column. To drain out that extra washing buffer we will centrifuge it at 7000 rpm for 2 minutes. So we are going to centrifuge it at 7000 rpm for 2 minutes now. Put this into the centrifuge machine, close the lid properly, set the time for 2 minutes and start the centrifuge machine. The centrifuge run is complete, open the centrifuge, take out our tubes. So if you see carefully some extra washing buffer at the bottom of the tubes. Earlier we did it for 2 times but still there was some washing buffer in the columns and it is drained in the next centrifuge. So we will pour it out, carefully take out the columns, discard it. So these are our columns and our plasmid DNA is in these columns. Now we will elute our plasmid DNA into new tubes. So we will transfer our columns into the new centrifuge tubes. We will take the new centrifuge tube, put our column into it. Now you can discard the previous tubes. So I have transferred our columns into new tubes and will add the allusion buffer. Please label our tubes according to the column numbers. This is number 1 so I am going to label it as number 1 and this is our number 2. So I am going to label it as number 2. Now I will add allusion buffer 50 microlitre each in each column. This is allusion buffer from the kit. I will take the yellow pipe it and we will adjust it at 50 microlitre. I will attach the yellow tip. This allusion buffer will dissolve the plasmid DNA into it and will pass through the column into the newly added tube. Discard the tip. Now we will put our tubes into the centrifuge machine and we will run it at 7000 rpm for 1 minute. Carefully close the lid. Now start the centrifuge machine. So our centrifuge run is complete. Now we will take it out of our tubes. You can see the allusion buffer has dissolved the plasmid DNA and it has passed through the column and it is at the bottom of the tube. And this is our extracted plasmid DNA. So now we can discard our columns and cap these tubes and use this plasmid DNA for further processing. So we will discard the columns. We will discard the second column as well. And this is our extracted plasmid DNA. This can be used for transfection and further any process. We should store it at minus 20 immediately and then after we can use it for any other process. Dear students, this was our plasmid DNA extraction practical. While doing this experiment, you should take care of these points. While the cell lysis solution step is done, do it quickly. Otherwise it will re-nature the genomic DNA and the genomic DNA will be extracted along with the plasmid DNA. At the resuspension and lysis buffer stage, do not vertex it vigorously. Otherwise it will re-nature the DNA and breaks it into the small fragments. And the third and the important point is that wear gloves and glasses to protect your hands and eyes. Thank you very much.