Extracting Plasmid DNA: How To Do a Miniprep

0:11 One of the smellier protocols you'll come across as a molecular biologist is the bacterial plasmid prep or the extraction of plasmid DNA from bacterial liquid cultures. Bacteria are little DNA factories. We can design plasmids or circular fragments of DNA that we insert into a bacterium and they will diligently replicate it to quantities that are useful for downstream applications like sequencing or cloning. But in order to do any of those downstream experiments, we first need to extract the plasmid from the bacterial cells. The most commonly used bacteria for this purpose is E. coli. Yeah, the one found in your poop. But don't worry, the E. coli that we grow in the lab are harmless, even if they do not smell very nice. Preps come in lots of different sizes,

0:56 depending on the volume of bacterial culture you start with. We've got mini preps, midi preps, maxi preps, mega preps. Giga preps! The chemistry and principal stay the same. You just need to change the size of the equipment you use, and of course, the larger the culture you start with, the more plasmid DNA you'll get at the end of the prep. The most common size you'll come across in the lab is the mini prep, which starts with just one to five mL’s of bacterial culture. So that's the one we’ll be demonstrating today. First, let's culture our E. coli. We'll need LB or Life Broth. Very yummy food if you're E. coli, with the correct selective antibiotic already added. In this case, my E. coli contain a plasmid that confers Kanamycin antibiotic resistance. So I have added Kanamycin to this LB. This ensures that only E. coli containing the plasmid will grow.

1:44 Any random bacteria that happens to fall inside the tube will be killed by the antibiotic. We'll also need culture tubes. They're sterile, so they won't be contaminated with other bacteria, and they have a loose fitting cap that allows airflow but prevents contamination from falling in, and make sure they're labeled with your sample info. I'm going to add three mL’s of LB to my culture tubes using a sterile pipette, and I want to close my culture tube and LB caps promptly to prevent contamination. Next, we need to inoculate the culture with some E. coli. You can get your bacteria from a frozen glycerol stock or from a colony on a petri dish. For either just take a sterile pipette tip and poke it into the bacteria and add it to the culture tube.

2:29 And finally, let's place the inoculated cultures into an incubator at 37 degrees Celsius. E. coli like it, nice and steamy, just like the inside of your intestines. And we're going to shake them at 200 rpm, so that the bacteria don't settle and form clumps and so they have equal access to the nutrients in the LB. And we'll leave them like this for 12 to 16 hours, so they can replicate to their heart's content. And after 12 to 16 hours they look like this. You can see that our culture has become nice and cloudy, a sign that our E. coli have been happily reproducing overnight and making tons of plasmid DNA in the process. And now it's time for the mini prep. You can either get a miniprep kit from a scientific supply company

3:17 that'll provide all of the tubes and reagents you'll need in a nice little box, or your lab will have recipes on hand for making their own reagents. The steps and general chemistry are pretty similar across the board, but make sure to read your protocol carefully before you start, because there can be slight differences in volumes and incubation times across kits or your lab might have special tips and tricks for their workflow. We're going to use a ton of test tubes for this mini prep. So first I'm going to get all of my tube labeling out of the way. I like to assign each sample a temporary number so I don't have to write out the full name on each tube, but I make sure to write down the key in my lab notebook first so I don't forget. For each sample I'll need three tubes;

4:04 a 1.5 mL tube for the first few steps, a tube with a column insert for the middle steps and another 1.5 mL tube for the final plasmid collection. I'm doing four minipreps today, so I'll need 12 tubes total. We'll explain what each tube is for as we go through the protocol, but for now I'll label the first two with a temporary number and the final collection tube with my full sample info and the date I'm performing the mini prep. While LB is delicious for our E. coli, it is not delicious for our mini prep, so we want to get rid of the LB and only work with the E. coli for the rest of the experiment. To do this, I'm going to pipette one meal of my culture into the first 1 mL tube and spin it in a centrifuge for one minute at 13,000 rpm.

4:49 The spinning separates the components of the culture by density. The dense E. coli will form a pellet at the bottom of the tube. So when it's done centrifuging, we can just dump the LB out and the pellet will remain. Make sure to dump it into a designated biohazard waste container, since it still has some bacteria in it. And I should mention now that all waste associated with this experiment, including tubes and pipette tips, should go in the biohazard bin. Our bacteria isn't much use to us in a clump though. So next we add 200 microliters of resuspension buffer

5:42 and vortex until the bacteria get redistributed in the liquid. This will allow the subsequent reagents we'll be using to have equal access to all the bacteria. The next steps go by quickly, so I'm going to show you the method and then explain what's going on chemically. First, I'll add 200 microliters of lysis buffer. Gently invert the tube a few times to mix it and start the timer for a one minute incubation. Then move on to my next sample. Add buffer, gently mix. But do not use a vortex to mix the samples. They’re really delicate at this point.

6:29 And you probably noticed that these buffers are really colorful. Miniprep kits often include color changing reagents, and they say it's for scientific reasons like, "knowing your reaction is proceeding correctly”. But I'm pretty sure it's also just to keep us entertained. But no worries if your kit or homemade buffers don't contain pretty colors, they’re not necessary for the mini preps to work properly. After one minute is up, we add 400 microliters of neutralization buffer to each sample. Again, gently inverting the tube immediately upon the addition of the buffer. A chunky precipitate will form and try not to get hypnotized by the color to change yellow, because this only incubates for 2 minutes. After 2 minutes, we'll spin these tubes in the centrifuge

7:16 at 13,000 rpm for 5 minutes. So that gives us time to explain what the heck just happened. Let's go back to when we added the lysis buffer. The lysis buffer contains a detergent which rips the E. coli’s lipid cell membranes apart, kind of like how dish soap attacks the grease on your pots and pans. All of the proteins and genetic material come spilling out of the bacteria and the detergent denatures the proteins. They unfold and become a tangled mess. Each E. coli cell has two types of DNA. The plasmid that we designed and also their own genomic DNA that contains all of the instructions about how to be an E. coli bacterium.

8:04 But we don't care about how to be an E. coli bacterium, we only want our plasmid. So how do we make sure that we only get the plasmid DNA and not any genomic DNA? Well, the lysis buffer also contains sodium hydroxide, which has a very high pH. It denature both types of bacterial DNA, by disrupting the hydrogen bonds between base pairs. So the double DNA strands unzip to become two single DNA strands. The selection for the plasmids DNA happened in the next step when we added the acidic neutralization buffer. The DNA molecules are able to zip back up again. The plasmids do it super easily because they're relatively small, only a couple hundred thousand base pairs.

8:49 But the E. coli genome is 4.6 million base pairs long and it can’t zip up as easily. So it gets tangled with all of the proteins and other cellular debris. The neutralization buffer also contains potassium, which will force the proteins to come out of solution, bringing the genomic DNA with them. So we're left with a bunch of solid cellular debris, proteins and genomic DNA, that was a solid precipitate we observed when we added the neutralization buffer, plus a liquid solution containing plasmid DNA. I should also mention that the neutralization buffer has some RNSA in it, which is an enzyme that degrades RNA. We don't want RNA either, only our plasmid DNA. And the pretty colors just tell us that the pH changes were happening correctly throughout the process.

9:37 The solution is pink in the presence of the base and turns yellow when we added the acid. Okay, now back to the present where our centrifugation is complete. All the solids, including the genomic DNA, are in the pellet at the bottom of the tube, and on top is the liquid solution containing our plasmid DNA. That solution may look nice and clean, but there are actually a lot of other things floating around in there with our plasmid DNA, like salts and leftover RNSA. The subsequent steps of the miniprep will wash all of those molecules away and just leave our DNA behind. We're going to use this little contraption. It's a tube with openings at both ends and a little column made of silica at the bottom, and it sits inside a collection tube. We’ll remove our solution from the 1.5 mL tube

10:23 being careful not to disturb the debris pellet and pipette it into the column. Then we centrifuge the column and collection tube together for one minute. The liquid will pass through the silica column, and as it does, the DNA sticks to the silica and everything else passes through. We can just dump the flow through. But it's possible that some residual molecules are still mixed in with our DNA.

11:09 So we're going to use two different buffers to wash the column. These wash steps vary a lot from kit to kit, so be sure to double check your instructions. The first wash buffer removes any remaining proteins, RNA and cell membrane components. We'll add 200 microliters to the column and centrifuge for one minute. Dumping out the buffer afterwards. The second wash buffer removes any remaining salts. If you're opening a new kit for the first time, you'll have to add ethanol to the buffer.

12:03 After you do, check the box on the bottle and add your initials and date, so the next person to use the kit knows you've already added it. Okay, now I'll add 400 microliters of wash buffer two, centrifuge, and dump the flow through. And now it's finally time to collect our squeaky clean plasmid DNA sample. First I’ll transfer the column to the final collection tube that has all the sample information in the label and double check that the number on the column tube matches my sample.

12:53 Then I add 30 microliters of elution buffer to the column. Make sure you pipette precisely on top of the column because this volume is pretty small. However, you don't want to poke the column with your pipette tip, so just be careful. I like to steady my pipette with my finger as I do this part. We let it sit for one minute. the elution buffer is basic, which will cause the silica in the column to deprotonate and become negatively charged. DNA is also negatively charged and the like charges repel each other. So the DNA will become unstuck from the column and flow through

13:39 into the tube with a buffer when we centrifuge, which we're going to do now for one minute. Hooray! Our nice pure plasmid DNA is in each tube now. Let's discard the columns and put the tubes on ice and use a spectrophotometer to tell us how concentrated each plasmid sample is.

14:36 Woohoo! There's 144.37 nanograms of plasmid per microliter of solution in this sample. That's amazing! The 260 / 230 and 260 / 280 values are quality metrics and should be around 2 and 1.8 respectively. Our sample is at 2.22 and 1.77, which is really great. Record all of your concentrations in your lab notebook, you'll need them for downstream applications. And finally, you can store your plasmid samples in the freezer at -20 degrees Celsius. Good night little plasmids, sleep tight.