Hi, I am Alex Hoge. I work in gym Hall Lab in the Department of Physiology and biophysics at University of California Irvine. And today I'm going to show you how to transform electrically competent e coli by the method of each shock.
So today I'm using this technique to transform a coli with my lation product because I'm doing a prob to do some whole mount ization in zebrafish. Okay, so we are going to start to do the transformation. So first you need to have your water bath or dry bus At 42 degree.
You need to have your bacteria that you just took out of the main city in ice. Today we use electrical competence cells from ities and also your DNA in ice. Also you need tube of SOC media at home temperature or 37 and your plates of LV plus antibiotic media.
So now I'm going to add the DNA with the bacteria. So I work next to the flame to not contaminate the bacteria. So I add lation with the bacteria.
It's 50 microliter of bacteria and then immediately back in the ice you can flick it a little to mix the bacteria and your DNA and you leave for up to 15 minutes in ice. So now 15 minutes have passed and we are ready to do the heat shock, which is to put the bacteria at 42 degrees for 45 seconds. So I take the tubes out of ice in directly at 42 degree set, 30 timer 45 seconds, and then I will put them back in ice very fast and put them out of 42 directly on ice.
And then we wait for two minutes. So now I'm going to add the SOC media into the bacteria and put them at 37 for 30 minutes. So I vape it 500 microliter of the SOC media and I put it into the bacteria and put them in my little makeshift chamber.
So if you are going to use eend tubes and makeshift chamber for your incubator, make sure to put your eend tubes horizontal because they will shake much better like that. So now we are ready to put the tubes in the shaker. So that why we have this little chamber at 37 degree for 30 minutes.
So now we have finished incubation at 37 degree and we are going to plate the bacteria on lb plus antibiotics. So in my case it's chloral. So at I 50 microliter of each sample and put it on the plate.
So with the remaining 500 microliter, you spin them down in a little tabletop, some refuse in order to peel it the bacteria. So after ification we get a nice pellet and what we are going to do now is remove almost all the SOC media, just leave 50 microliter and then we can plate this 50 microliter of concentrated bacteria. So I pay paid approximately 450 microliter out.
I just leave this much. So now I'm going to suspend the pallet in the 50 microliter left of SOC media. And after that I can plate this 50 microliter onto, onto another plate.
So I resist each pallet, then I puppet it and I plate it. So there will be at the end two plate per sample. So now we have to spread the bacteria onto the LB plate.
So I'm going to use some autoclave glass beads, but you can also use a pipe past that you shape into spreader like that. So I'm going to put few beads onto the Petri dish. I like that.
Well, 10 15. So after you add the beads, you put all your plates in a pile like that and you gently shake them to have the beads spread the bacteria everywhere evenly on the plates. So as I move the plates until they're dry.
So, which means that you should not see big streaks of liquid when you move with the beads. For example, in that plate, the beads tend to collapse together and see a lot of streaks. It's sweat that one, the beads move freely, that one is dry.
So now the plates are dry so we can discard the beads. So I just go the pull like that so you can recycle them. So now we are going to put the plate in the incubator at 37 overnight, and you put the plates upside down after 12 hours of incubation at 37, we get the plate out of the incubator.
And as we can see, we have fewer colonies in the plate where I plate it 50 microliter than in the plate where I plated the rest. What is important when you transform bacteria is to have the right amount of colonies on your plate, the right density. So in this place you have very few colonies, but if you want more colonies, you can have the good example in this plate where it's exactly the right density, approximately 100 colonies per plate.
This plate is a bad example because there is too much colonies and you cannot pick individual colonies. So I just showed you how to transform e coli by each transformation. So the very important aspect of this technique is to keep everything or ice cold before the shock.
Then 45 seconds normal, more at 42 degree back in ice. And then everything else has to be at a warm temperature or 37 degree. So now I'm going to screen my colonies by PCR screening.
So one of the point also is to have two plates. So one, we expect to have a lot of colonies and those one less colonies. And what is important is to have your plates very dry with the use of beads or the pipe past.
Thanks for watching and good luck with your confirmation.
