Size Selection and Cleanup with NEBNext Ultra II and SPRI beads
This video walks you through the size selection and cleanup steps using the NEBNext Ultra II DNA
Size selection enriches for molecules that were sheared to the desired size and have an adapter ligated to each end. Size selection is accomplished using magnetic beads. There are two rounds of selection: the first removes DNA fragments larger than the desired size, and the second removes DNA fragments smaller than the desired size. This is accomplished using specific ratios of the bead solution to total volume, and the volume of beads required varies depending on the desired fragment size. Magnetic beads should be used at room temperature and they should be vortexed before use. For the first round of size selection add vortexed beads to the reaction and mix well by pipetting up and down at least 10 times. Incubate for five minutes at room temperature, then place the reaction into the magnetic field. After approximately five minutes the beads should have separated from the supernatant. Carefully remove the supernatant and avoid disturbing the pellet. Vertical bar magnets pull the beads to one side of the tube or well, and the supernatant can be removed by placing the pipette tip on the opposite side of the tube or well from the magnet. Plates with circular magnets pull the beads uniformly to the walls of the tube or well, so the supernatant can be removed by pipetting straight down into the bottom of the tube or well. Being careful not to touch the sides. In this first round of size selection the unwanted large fragments are bound to the beads, and the desired DNA is in the supernatant. Retain the supernatant. For the second round of size selection add new magnetic beads. Incubate for 5 minutes. Expose to a magnetic field, and remove the supernatant.
Discard the supernatant as the desired DNA is bound to the beads in the pellet. Watch the beads with 200 microliters of eighty percent ethanol while they are in the magnetic field. Incubate for 30 seconds at room temperature and then remove the ethanol. Repeat this wash step once. Allow the beads to air dry while the beads are in the magnetic field, but avoid over-drying the pellet. Remove the tube or plate containing the dried pellet from the magnet and elute the library from the beads by adding 17 microliters of Tris Hydrochloride or 0.1X TE buffer, followed by mixing well by pipetting up and down or vortexing. A quick spin may be required to collect the liquid to the bottom of the tube or plate. Incubate for an additional two minutes at room temperature. Return the sample to the magnet for five minutes or until the sample clears and remove 15 microliters of the supernatant. Add this 15 microliters to a fresh PCR tube for library amplification. This step also uses magnetic beads, but this time there's a single step after which the desired DNA, the library, is bound to the beads. Don't discard the beads. Add 45 microliters of vortexed magnetic beads to the reaction and mix well by pipetting up and down at least 10 times. Incubate at room temperature for five minutes followed by exposure to a magnetic field. After five minutes remove and discard the supernatant without disturbing the bead pellet. Wash the beads with freshly prepared eighty percent ethanol as previously demonstrated. Allow the pellet to dry.
Remove the tube or plate containing the dried bead pellet from the magnet, and elute the library from the beads by adding 33 microliters
of 0.1X TE buffer, followed by mixing well. After a quick spin, incubate at room temperature for 2 minutes. Return the sample to the magnetic field until the sample clears, approximately five minutes, and remove 30 microliters of the supernatant containing the library to a new tube.
This library can be stored at minus 20 degrees Celsius.