how to transform E coli by heat shock

you you everybody welcome back to synthetic biology 1 today we're doing heat shock transformation of e.coli it's been about 10 minutes these cells have thawed and they are now ready to use so first I will need to take an aliquot of these cells so I've got about 200 microliters of cells here total but I only need 20 to do my transformation so I will take my aliquot here don't forget to label okay I'll put that on ice let that cool for a couple of minutes and then I'm going to aliquot 20 microliters of competent cells into that tube so we're keeping everything cold for this okay so there's my 20 microliters of cells to this I am gonna add just a half a microliter of the plasmid that I want to transform so in this case we're transforming an intact plasmid at a very high concentration so I only need to add a tiny tiny amount to the cells for successful transformation if I was working with like a ligation product for example I would use a larger volume maybe two or three microliters to get to get the number of Transformers that we need but we for this we just need a just a whisper whisper of cells so we'll go all the way down to half a microliter and that to our competent cells very gently so you'll notice that I'm pipetting the plasmid directly into the cells right so when you when you're working with a volume that small you really can't afford to lose anything so you pipette directly on top of the cells okay now I'll mix this by flicking so I give it just a little a little flicking motion and then tap it down now you want to leave that for mmm five minutes ten minutes just to let the plug give the plasmid a chance to stick to the cells I don't really know why but people say it improves the transformation efficiency okay so we're back it's been 10 minutes these cells are well mixed now and they are ready to administer the heat shock so for the heat shock part of the heat shock transformation I'll be using this heating block you might not use a heat shock you might use a water bath or some other way of raising the temperature to 42 degrees Celsius and we're gonna do it for exactly thirty seconds so I don't I don't have a timer handy around here so I'm just going to time it on my iPhone okay so move 30 seconds on the heat block alright that's 30 seconds now we take it out go immediately back to the ice and we'll leave it two minutes on the ice to recover so while the cells are recovering we can prepare the media that we use for the next step of the protocol so I'm gonna recover these cells in plain elbe media so this is this is just standard ecoli lb media other transformation protocols use enriched media like you might see of SOC for example and in some cases a richer media can improve the recovery of the cells improve the transformation efficiency but I find that plain old lb works works perfectly well for most applications so that's what we're going to be using okay so that's two minutes on ice now I'll take the cells out and add 200 microliters of plain lb so this is a ten to one ratio we're using 200 microliters of lb to recover 20 microliters of competent cells okay so again we'll mix that a little bit by flicking it and now we want to recover these cells at 37 degrees C for 15 minutes to an hour depending on the plasmid that you're transforming and the antibiotic resistance that you're using so the the purpose of the recovery step is that it gives the cells a chance to grow a chance to recover from the calcium chloride treatment and to express the antibiotic resistance marker that's contained in the plasmid okay so we want to grow the cells at 37 degrees C for half an hour with shaking to create the optimal conditions for cell growth so there are quite a few ways that you can do that I'm going to I'm going to show you my way so I'm gonna I'm gonna incubate these cells in the in a regular incubator using what I call the sandwich method so I'll take another another rack like this one tape the two racks together to make a kind of sandwich so now the now the tube is is horizontal and so this gives the cells a lot of room to shake around when they go on the shaking incubator it works for me okay so we're back the cells are all recovered the sandwich worked in this case we were we're using a chloramphenicol resistant plasmid so we've recovered the cells for a full 60 minutes in the incubator okay so these cells are recovered and ready to use and all that's left is to plate them so over here I've got some plates of l8b agar with the appropriate antibiotic in this case chloramphenicol and I'm gonna prepare two plates one with 200 microliters of cells and one with 20 microliters of cells so the reason that I like to prepare two plates is I don't know exactly what the transformation efficiency of this plasmid is going to be and this improves the odds that I'm gonna get exactly one plate with the right number of colonies okay so to plate these cells I'm gonna use these so these are glass beads that I'll add to the plates and they have the effect of spreading the media evenly around the plates without absorbing too many bacteria and so they're gonna they're gonna give us a nice even distribution of bacteria on the surface of the plates okay so plate number one gets 200 microliters and plate number two gets 20 microliters shake them turn turn okay that's going to give us a beautifully even distribution of bacteria on our plates toss the beads and that's it these are ready to go in the incubator overnight so here I have an example plate that I prepared yesterday and as you can see this is after about 16 hours of growth I have these nice beautifully formed colonies of transformed bacteria using the heat shock transformation protocol

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Id: 2ligY8VOzz4

Length: 11min 13sec (673 seconds)

Published: Tue Jan 12 2016