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DNA extraction

DNA needs to be extracted from biological samples before further analysis. Watch Forensic Analyst Federica Giangasparo explain more.
Hi, Federica. Hi, Gabriella. Hello. This is Natsumi. Hey, nice to meet you. Nice to meet you. We’ve got these two swabs, could we do DNA profiling? Sure. I’m the DNA analyst for King’s Forensics, so I will be more than happy to walk you through the steps. OK. Perfect, I’ll leave you guys to it. Enjoy your time, Natsumi. Thank you. Bye. Right, let’s place them in the rack. And, Natsumi, if you want to have a look at the ID of the samples Gabriella provided– OK. –and maybe start unscrewing the cap, please.
The method we select to do today– there are several methods we can use for DNA extraction. But the method we use today is a semi-automatic system, which is the easy one via robot. Of course, contamination is one of the major issues we need to be careful of when working in a forensic lab. Yes. So everything has been treated by decontamination via UV light. I see. So, first of all, we’re going to use our reagents and we’re going to need an extraction negative as well, so that everything would be treated in the same way for the sample and for the negative. OK.
The buffer I’m just adding now is a lysis buffer, so it will help the cell to burst out and to allow the cells to liberate all the DNA that contains within the cell. OK. To improve this method, we’re going to use also proteinase K, which is an enzyme which is designed to break the proteins– they’re forming the phospholipid membrane. So we’re going to add the enzyme and we’re going to gently mix and gently mix again.
And we did the same for the extraction negative. So it is important to run an extraction negative together with the actual sample just to assess that the extraction is working correctly and we don’t have any contamination within our reagents. I see. So, now that we have mixed the buffer lysis with the proteinase K, we’re going to put it on an incubation at 56 degrees, which is the optimal temperature of function of this enzyme– proteinase K. OK.
But before doing that– because we want the burst of the cell to be the most optimal as possible, we’re going to vortex the samples as well. Oh, OK. [MACHINE HUMS]
It’s a mechanic action that will help the process of extraction. I see. [MACHINE HUMS] Now, we’re going to place the tubes inside the terminal block at 56 degrees– just checking my names and my extraction negative. We’re going to start the reaction and wait for at least 15 minutes to half an hour. In order to make the process even more optimal, we can transfer the tubes to 100 degrees after about half an hour just to make sure that everything is degraded and everything is burst out in solution. So we want the nucleic acid free in solution.
So, we have done our incubation step at 56 degrees, so the optimal temperature for the enzyme to work– the proteinase K, and we moved it down to 100 just to optimise it and then centrifuge it. So, now, we’re ready to move the samples to the actual extraction tube. OK. Then we’ll be ready to go to the robot straight away. So, first of all, we need to check the labelling. So, label number one will go with sample number one, and label number two will go with sample number two. Yes. And the extraction negative will not be needed to be transferred.
So we’re, now, ready to load the samples onto the robot. And, Natsumi, can you please pass me the tube– Sure. –and the tube holder? This one?
Thank you. So I have here prepared what I need for the robot to work.
So the robot works isolated DNA through beats isolation. So this is how a cartridge normally look like. So the black part is the beats and then we go through several steps. One of the steps is lysis, and a couple of washes and, finally, it will elute in water. So we’re going to prepare one cartridge per sample we are analysing. Finally - and on the robot, it will have another heating block over there, and this blue bar is the magnetic bar that will help through the separation once the beats have linked with the free nuclease solution.
So we put the cartridge here, so we’re going to have the samples on here. The robot is really helpful, so it guides you through it.
Right. So, now, the number of cartridges, the dilution sample– so, again, taking into account that number one– sample number one, sample number two. To finish the extraction negative, the tube and tube holder up here and here, and the final thing is for the sample that we loaded before. So we’re, now, ready to press start and at the end of the process - which lasts normally about 20 minutes - we’re going to have 50 microlitres of really pure DNA extract that we can use for the amplification step. OK.

DNA needs to be extracted from biological samples before further analysis. Watch Forensic Analyst Federica Giangasparo explain more.

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