Hi! Today we'll be extracting plasmid DNA from bacteria, it's a protocol is usually called a miniprep It's called minipreps because we will be extracting DNA from a small volume of bacterial culture. You can also do midipreps and maxiprep from bigger volumes, but we won't discuss it today So to start you have to start a bacterial culture for this we need a culture tube and some media To keep it sterile we will be working around the source of heat I will now pour some media into the cell culture tube About five mL is enough We also need to add antibiotics to the medium. In this case it's kanamycin The antibiotic is needed to prevent other contaminants from growing this way only our plasmid our bacteria containing our plasmid will grow in this medium And now we have to pick one colony one single colony from a plate. We also are doing this in sterile environment So now all we have to do is to label our tube and put it to incubate at 37 degrees overnight So now our culture has grown and we are ready to prepare the miniprep actually the best way is to take a 2 milliliter tube and Extract plasmid DNA from 2 milliliter of bacteria, you don't have to be in sterile environment for this step so I just poured the 2 milliliters into the tube And we will now have to centrifuge it around 2 minutes, 2 to 3 minutes at 6000 RPM to avoid damaging the cells don't centrifuge too fast I'll actually use another tube of two milliliters to equilibrate the centrifuge And it will centrifuge the cells to keep all the bacteria on the bottom of the tube in the pellet So our pellets are ready. They are here in the bottom of the tube, and we're now ready to continue with the mini prep For all the next steps I will put gloves to protect DNA contained in the samples from the DNase so that could be on my hands So now we have to discard the supernatant by just pouring it out, and we only keep the pellet on the bottom First step is to add cell lysis buffer Usually for minipreps you'll be using a commercial kit and all the solutions already come prepared for you The kits differ a little bit between the brands, but the principle is the same. First we adding the cell resuspension buffer 200 microliters of this buffer And the point is to resuspend the cells in this solution So we pour it into the Tube and then You can either gently pour the solution or the easier way Is to do the following, and then the pellet is resuspended Next step we will add the cell lysis buffer. This will pour the membranes open, it will disrupt the cell Membrane and the cell contents will pour inside the solution so 250 microliters of the lysis buffer And we have to mix it gently to prevent disrupting the DNA The next step is to add the neutralization buffer It will neutralize the ph and would also cause chromosomal DNA to precipitate and only the plasmid DNA will arrest in the solution 350 microliters of neutralization buffer and you will see that the solution becomes troubled and the precipitate appears We also mix the tube gently to avoid disrupting the DNA Now we will have to separate the separate the precipitate from the solution For this will perform another step of centrifugation This time we'll centrifuge at higher speed. Actually you can use the maximum speed of the centrifuge for this So in our case, I will centrifuge for five minutes at fourteen thousand six hundred RPM So now our pellets are ready, and we can continue as you can see centrifugation separated very well the solution from the pellet and in the pellet we'll have all the lipids and the Membrane and the chromosomal DNA all precipitated on the bottom and now we're going to proceed with the for the next step for this we'll need a collection tube and The filter column they are usually provided with a kit We will have to pour the supernatant on top of the column So the supernatant now contains our plasmid in the solution You can now discard the pellet and proceed And proceed with a with a column We will now centrifuge the column one minute at maximum speed To let the solution flow through the filter Now that we have let the flow through get through the column we can discard The flow-through and replace the same column into the same collection Tube. We will now wash the filter with a wash solution containing ethanol This will keep the DNA bound to the column, but it will allow the rest of the contaminants go into the waste tube down here So we'll wash twice with 500 microliters of wash buffer Once again, we centrifuge one minutes at maximum speed So we now repeat the wash with the same amount of wash buffer So once again we discard the flow through We replace the column And we have to add the wash buffer So now once again. We discard the flow-through and Now I'll have to Dry the column to avoid all the ethanol present For this, we'll centrifuge two minutes at maximum speed without any solution added to the column Good. At this step it's essential to make sure that there is no ethanol left in the tube So we just keep the column. And if you want to check if there is still ethanol left You can flick the tube a little bit with your fingers There's no droplets left that means that the column is ethanol free we can now take a clean 1.5 mL Tube in which we will collect our DNA So place the filter on top of the tube You can wait a minute or two just to make sure that all the residual ethanol evaporates or it can go straight ahead So to collect our plasmid DNA we will now have to add the elution buffer or you can use just a nuclease pure water nuclease free water The volume to add depends on the concentration you want to get into in the end. If you want a really concentrated sample you're interested in putting as little water as you as you can. The minimum volume is about 35 microliters, but you can get up to 100 microliters if you don't care about the concentration so much. I usually do something in the between So I will put 40 microliters of nuclease free water At this step it's important to pour the water right in the middle of the of the column and not to touch the filter with the tip So I pour the water and now we keep the column like this for a couple of minutes to wait until the water soaks the filter and all the DNA comes from the filter into the solution Now we can elute the DNA into our clean tube For this once again, we will centrifuge one minute at maximum speed so at this point our protocol is finished and you can discard the column and You have your DNA miniprep. Now the only thing that's left is to label the tube correctly So I will put the name of the plasmid and the date the best way to label is to put the name of the plasmid both on the tube lid, and on the side. And now we can check the concentration of the DNA by a spectrophotometer.
