[Music] hi welcome back to synthetic biology 1 today we're going to be preparing competent cells so how I like to start is by preparing my solutions so we have two solutions that we need to prepare one is a hundred millimolar solution of calcium chloride and another one is 100 millimolar calcium chloride with 15% glycerol so we'll need one and a half grams of calcium chloride two times [Music] all right and let's not forget to label our solutions so this guy is gonna be our 100 millimolar calcium chloride and over here 100 millimolar calcium chloride with 15% glycerol ok so label these and I'm being careful to label these bottles near the top because we're gonna put them on ice and if the labels get wet they can fall off ok so now I'm gonna take my water and I'll go ahead and add it to the calcium chloride solution bringing the total volume of the solution up to 100 milliliters just pour that in top it off at 100 milliliters I'm also going to add some water to the calcium chloride solution but I'm not gonna add the full 100 milliliters yet I'm just going to take it up to 50 mils so that there's room left over for adding the glycerol okay so now let's pipette the glycerol I need 15 mils of glycerol and for this I'm using a hundred percent glycerol solution and it can be really a pain to pipette so I'll have to be extra careful one tip here is just put the tip of the pipette into the glycerol because otherwise the entire pipette will be coated with glycerol to be a little patient to get exactly the right level let the excess glycerol drip off and now this is why I added some water to the solution because when I when I add the glycerol I want to pipette up and down just to wash the excess glycerol from the pipette tip okay cool and now we'll take the water and just top off the solution bringing the total volume up to 100 mils you can you can tell I'm not being too careful with my measurements for these solutions and that's okay I don't need to be there they're quite forgiving and close is good enough okay there's my two two solutions prepared mix them up and I'll store these on ice so you want to store them on ice so the most important thing about preparing competent cells is that once we've resuspended the cells in the calcium chloride solution we want to keep them always ever after at four degrees Celsius because if we allow the temperature to come back up the cells might start to lyse okay so that's our solutions now we're going to go ahead and start the culture of cells that we're going to actually be making competent so for this I have a plate of mg 165 5 you might want to use nab turbo or dh5 alpha or some other cloning strain this is just a wild type strain of e. coli and it's it's what I happen to have ready around the lab okay so we're going to start out five mill culture of e. coli in LB media so these are 14 male Falcon tubes don't forget to label see there's my five mils and now I am going to take an inoculation loop and pick just a single colony of e.
coli from my plate to start my culture with it's important when you make competent cells to always start from a single colony of e. coli because that way you know that the cells are genetically homogeneous and not contaminated okay so now I'm going to pop this in the incubator and grow it at 37 degrees with shaking overnight eight to twelve hours okay so it's eight to twelve hours later and my culture of e. coli is nicely saturated so the next morning when you're ready to prepare you're competent cells when you come in what you do is you'll take these cells and we're going to dilute them by a factor of 200 into a fresh 50 milk culture so we always want to make competent cells using freshly growing log phase e.
coli because they're the healthiest and they'll be the most competent so for my my 50 ml culture I'm using a 250 mil flask it's important to use a much larger flask than the volume of cells that you're growing so that the cells are nicely mixed and well aerated so they can grow quickly now to my 50ml of lb media I'm adding 250 microliters of my saturated e. coli solution okay I mix that around so we'll grow that in the incubator at 37 degrees for two or three hours until the OD reaches 0. 5 or 0.
6 which indicates that the cells are in the mid log phase and ready to become competent it's two or three hours later and I've collected my cells from the incubator and they're at an OD of exactly 0. 5 and so they are ready to go for the next step of the competent cell protocol so I'm going to take my my culture of e. coli and my freshly log phase e coli and I'll add it to a 50 ml Falcon tube like this and then put it in the centrifuge and spin it down at a maximum speed for ten minutes to produce the pellet of cells for the next step in the protocol okay so now we've collected our pelleted cells let's turn them into competent cells by adding calcium chloride solution so the first thing that I'm going to do is very quickly pour off the supernatant from the centrifugation store these cells immediately on ice so from now on everything that touches these cells should be at four degrees Celsius or colder now we're going to resuspend these cells in our calcium chloride solution here 15 mils okay so i'll resuspend the cells by pipetting up and down the pellet should come apart pretty easily after a few rounds of pipetting giving us a nice uniform resuspension of cells we don't want to spend too long doing this or the cells will start to warm up okay now I'm going to leave these cells on ice for at least four hours or it could be longer even overnight okay so after the second round of centrifugation we will treat the cells the same as before except now we are going to resuspend them in four mils of the calcium chloride and glycerol solution so I'll go ahead and pour off the wastes and resuspend in four mils of calcium chloride glycerol okay so now these cells are ready to use they're ready to transform or to store however you want for the final step I'm going to aliquot this large culture of cells into smaller eppendorf tubes that will be more convenient to transform so for that I am going to need even more ice and I'm using this aluminum rack to cool the cells so this will transmit the heat very rapidly from the tubes to the ice and keep everything nice and cold so I like to aliquot into these 1.
