How to Handle Magnetic Beads During NGS Library Preparation

[Music] welcome to this tutorial on next generation sequencing we'll be reviewing the handling of magnetic beads in ngs library preparation [Music] bead cleanups purify nucleic acids and remove any unwanted contaminants such as salts primer dimers and dntps [Music] nope that's too much manual work let's get automated here on an ep motion bead cleanups can be tricky if handled manually so we'll get help from a robot we'll be taking a closer look at what happens in each well of the sample plate in this process [Music] the first step is the binding of the beads to the sample the volumetric ratio of beads to dna sample will influence the length of the dna fragment recovered let's play around with that ratio if we start at a 0.5 x ratio of beads to dna the larger fragments are selected for while smaller fragments will be removed during later wash steps here we lose a lot of the total fragments when moving to a 0.8 x ratio of beads to dna there is an increase in the number of smaller fragments selected for this can also include unwanted tiny fragments such as primer diapers the ratio of 0.65 x gives a tighter size distribution selecting fragment sizes needed for sequencing move on to the next step after the incubation of beads and sample the cleanup process begins the sample plate goes on to a magnet where the dna bound to the beads is separated from unwanted contaminants let's take a closer look [Music] if the sample vessel does not have a good fit with a magnetic plate some of the sample can be lost for all of the sample to be recovered it is important to have an optimized fit the supernatant can then be removed without disturbing the magnetic beads [Music] with the sample plate still on the magnet the next step is the ethanol wash first the supernatant is removed the ethanol is added to the pelleted sample and removed the pellet will then have to dry to remove any remaining ethanol all right we already managed to get pretty far well the ep motion did the job let's move on to the drawing step okay now we take a closer look at this step it is not as trivial as it might sound we'll look at three different scenarios the left pellet is air dried for a rather short time the pellet in the center is air dried for a couple of minutes the right pallet is dried on a heat block also for a couple of minutes all right let's resuspend and elude our first pellet [Music] after resuspension there is still a remaining ethanol that could inhibit enzymatic reactions downstream in the ngs workflow the pellet was too wet maybe we can get better results if we assist the drying process with heat oh now this pellet has been overdried the beads are brittle and it's hard to remove the dna this pellet was too dry as you can see here the pellet has been patiently air dried and the dna detaches easily during resuspension and illusion in the final step the dna is eluded from the beads the magnetic beads are then pelleted again the purified dna sample is transferred to a new well and is ready for the next steps [Music] we have summarized the most important points in this infographic to help you get the best results in your ngs speed cleanups thanks for watching this video the infographic shown is part of eppendorf's series of stay informed infographics available for download at eppendorf.com stay informed

Channel: Eppendorf

Views: 412,370

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Id: WBJTilok2xM

Length: 4min 56sec (296 seconds)

Published: Wed Mar 24 2021