Size Selection and Cleanup with NEBNext Ultra II and SPRI beads


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.

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