DNA Transformation into Bacteria

hi my name is Anne I'm a graduate student at the universe and today I want to talk to you a little bit about the molecular biology technique called bacterial transformation in our molecular bio labs what we're going to do is first we're going to mix some cells and some DNA together now our cells are in this tube you see this is a very small volume and we use these pipettes that can suck up small volumes and move around that we couldn't normally do with a regular so here myself you have to keep them on ice the entire time keep them stable put it into a secondary to next going to that also very small two together and store anonymous and wait 30 minutes hi again it's been 30 minutes and now we're going to put our cells DNA mix together it on a heat block 10 42 degrees Celsius so 42 degrees Celsius is the optimal temperature where the DNA literally gets sucked into the bacterial cells because the bacterial cell membranes are now weaker at this temperature so just for a little while we'll keep that on the heat lock and then we'll show it on ice so literally has to be just a few seconds and I love the member I get first or so ourselves have been next step is to put ourselves at 37 degrees C for an hour to grow and they need food to grow and our growth media right here going to add into ourselves now in our weight so let's review we've just done in the lab so in step one we mix our DNA coli cells my eyes are you Cola is special because it's not harmful to humans it's been mutated so it's not the typical type of e.coli they hear about the news it's not bad for you so we have the song ice with the cells and DNA they fix together for 30 minutes here in this little cartoon that within that time the bacteria the DNA can literally find each other and get closer next in heat shock at 42 degrees Celsius this is a special temperature because as you can see our little Ebola is not quite happy and it's cell membranes now become porous meet Lydia some scientists call and at that temperature the DNA rushes into the cell then we chillin at zero degree Celsius and ice for a couple minutes and then the membrane of the equality's restorer and your plasmid is inside so in our next step basically want to make more copies of that we don't want to just have one e.coli with one plasmid one of a bunch of e.coli with a plasmid what we do is we grow them at 37 degrees C for one hour and they duplicate every 20 minutes what happens that 20 minutes one goes to 2 goes to 4 4 goes to 8 and soap so in our next step this is our selective pressure step so our DNA is very important to have antibiotic resistance on it that is what creates a selection for T are you cooling when we grow them because we're going to have a mixture of e.coli as you can see here that had do not have a plasmid in it and one that do and we need to select for that so we grow the overnight on a plate with nutrient rich media on it and antibiotics which normally kill a colon bacteria and die by and then after that time period at 37 degrees C you'll have samples across ends it don't and along the Dubrow are the ones that have the DNA inside of it so it's then one hour at 37 degrees Celsius for ourselves so they warm they've been growing now we want to do is we want to select for the cells in here that have the plasma inside of it so you probably will drink why label right now we want to spread ourselves onto this media here without growing all sorts of other types of cells on there causes bacteria everywhere I want to make sure that we're on the growing the end of this spreader is now sterile a fraction of these cells so now we have a plate once everything absorbs we will grow at 37 degrees Celsius overnight in then check for the next day see we've columns growing here we have our plate with our bacterial transformation and now we're going to grow up overnight at 37 degrees seeing in an incubator yours are getting better hi fluor night before these colonies so it's been a day let's check on our bacterial transformation expected you do have colonies so now I'm going to show you how we're going to finish our experiment so we've gotten to the point where we've grown up colonies on these plates these very rich planes that have antibiotic resistance on them what they've done is that they select before the DNA that was put inside of it DNA plasma now this DNA is important not only for the antibiotic resistance that it allows for the eco lights who want to grow on these plates but also that it encodes for a particular protein of interest now that protein of interest is called GFP also known as green fluorescent protein now we're going to do is we're going to make the e coli make more of that protein and how we do it is that we take one of these colonies and we'll spread it on to one of these six well plates here that has an additional small molecule added into it if the LB which is the nutrient media kanamycin which is the antibiotic and the iptg which is the small molecule that causes the coli to want to make the GFP protein and this is what you'll be doing in your classroom so now I'm going to show you what you guys are going to do in your classroom so what you'll do is you'll take a sterile toothpick out of a container only hold one end of it the sterile side is the one that you're not touching you're going to use that side to pick up a colony any colony you'll do gently press against the blade get up you don't need a lot then you're going to open up the six wheel container and each person's going to get their own well and then you can spread it on to the world then throw the toothpick and the biohazard waste and what you're going to do is you're going to basically have your plates at room temperature for several days and then you're going to see what happens well hi everyone again I am at the University of Pennsylvania and I want to thank you all for listening to my talk on bacterial transformation I look forward to meeting you at your school and working with you

Channel: ejplab

Views: 39,598

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Keywords: EJPLab, Outreach, Videos

Id: iDfEVhePDPM

Length: 8min 27sec (507 seconds)

Published: Mon Dec 26 2011