 Dear students, today we are going to perform preparation of chemical competent cells. This is one of the methods of cloning, which is transfer of DNA from one organism to another organism. The purpose of this experiment is to make the cells more competent or more absorbance to the foreign DNA so it can be transferred inside the bacterial cells. For doing this experiment, make sure all the equipment are spliced and you have to perform the experiment using solution techniques. We need an overnight culture of E. coli and 150 ml lb broth, 50 ml calcium chloride solution for this purpose. I will perform the experiment in the biosafety cabinet and you need to follow me. Now we have an oculated 100 ml of lb broth with 0.1 ml of overnight grown E. coli culture. We need this culture to be put on shaker incubator at 37 degrees centigrade for about 2 hours and after 2 hours we need to check its optimal density so that it reaches about 0.5-0.7. When the od reaches about 0.5 and the cells start growing, we need to pellet down the culture. For this first we transfer the culture from plastic into the centrifuge tubes and then put into the centrifuge machine. After transfer of culture to the centrifuge tube, we need to centrifuge the culture. Second speed is 3000 G which is equivalent to 4000 rpm for this machine. Temperature 4 degrees centigrade and the time for spin is 5 minutes. As you know when we use centrifuge machine, we need to put always in a balance. So to balance the culture, I have put a balancer and start the machine. After centrifugation of the culture at 3000 G for 5 minutes at 40 degree centigrade, we will obtain supernatant and pellet. We need to transfer the supernatant to the discard and we need this pellet. To the pellet we will add calcium chloride which is 50 millimolar solution and this should be ice cold. The centrifuge tube as well as the solution should be ice cold. After complete pre suspension of the pellet, we will put this culture in ice for about 10 minutes and then centrifuge again to obtain the pellet. Bacterial suspension in a calcium chloride solution after incubation needed to be pellet down. For this we will centrifuge this 3000 G 4 degree centigrade and 5 minutes. After centrifugation of the calcium chloride containing culture at 4 degree centigrade 3000 G, we will again obtain supernatant and pellet. Now we need to discard the supernatant and we will dissolve this pellet in a calcium chloride solution to ML. After complete resolution, we will aliquot this suspension as our competent cells will be ready. One precaution is that we need to maintain 4 degree centigrade or ice cold throughout the experiment. So all the solutions, all the tubes should be ice cold and we need to handle the cells very gently. So when we shake this solution for the solution of pellet, we should be very gentle. It should not be harsh. Competent cells has been prepared with gristrol. We can store these competent cells at minus 80 degree centigrade for few months.