Add 40 μL of well mixed AMPure XP Beads to each well containing sample. Mix thoroughly by pipetting.
Incubate the plate for 5 minutes at room temperature.
Place the plate on the magnetic stand for 5 minutes at room temperature or until the supernatant appears completely clear.
Remove and discard all of the supernatant from the plate taking care not to disturb the beads.
With plate on stand, add 200 μL of freshly prepared 80% ethanol to each well without disturbing the beads and incubate the plate for at least 30 seconds at room temperature. Carefully, remove and discard the supernatant.
Repeat step 5, for a total of two ethanol washes. Ensure the ethanol has been removed.
Remove the plate from the magnetic stand and let dry at room temperature for 2 minutes or until the bead pellet is dry.
Resuspend dried beads in 51 μL of Resuspension Buffer. Mix thoroughly by pipetting. Ensure that the beads are completely rehydrated and resuspended.
Incubate resuspended beads at room temperature for 2 minutes.
Place the plate on the magnetic stand for 5 minutes at room temperature or until the supernatant appears completely clear.
Transfer 50 μL of the clear supernatant to a fresh well.
Add 40 μL of well mixed AMPure XP Beads to each well containing sample. Mix thoroughly by pipetting.
Incubate the plate for 5 minutes at room temperature.
Place the plate on the magnetic stand for 5 minutes at room temperature or until the supernatant appears completely clear.
Remove and discard all of the supernatant from the plate taking care not to disturb the beads.
With the plate on the magnetic stand, add 200 μL of freshly prepared 80% ethanol to each well without disturbing the beads and incubate the plate for at least 30 seconds at room temperature. Carefully, remove and discard the supernatant.
Repeat step 16, for a total of two ethanol washes. Ensure the ethanol has been removed.
Remove the plate from the magnetic stand and let dry at room temperature for 2 minutes or until the bead pellet is dry.
Resuspend dried beads in 32 μL of Resuspension Buffer. Mix thoroughly by pipetting. Ensure that the beads are completely rehydrated and resuspended. (take 1µl from the samples while on the magnet rack for qPCR dilution)
Incubate resuspended beads at room temperature for 2 minutes.
Place the plate on the magnetic stand for 5 minutes at room temperature or until the supernatant appears completely clear.
Transfer 30 μL of the clear supernatant to a fresh well.
We recommend quantifying your library with a fluorometer and checking the size using an Agilent Bioanalyzer. If on the Bioanalyzer trace there are two bands, one of expected size and one of higher molecular weight, a portion of your adapter ligated inserts have annealed to each other forming a bubble product. This occurs due to the long adapter length and is more prevalent when there are too many PCR cycles. This type of double band will not affect your sequencing results as the double stranded product will be denatured prior to cluster generation. As an extra verification step, a portion of your product can be denatured manually by heating the sample to 95°C for 5 minutes and then placing it on ice. The denatured product should appear as a single band on a Bioanalyzer RNA Pico 6000 Chip Kit.
qPCR is recommended to quantitate DNA library templates for optimal cluster density. This can be performed using any qPCR quantification kit with the NEXTflex Primer Mix.
Non-multiplexed libraries can be normalized using Tris-HCl (10 mM), pH 8.5 with 0.1% Tween 20. For multiplexed libraries, transfer 10 μL of each normalized library for pooling in the well of a new 96 Well PCR Plate. Mix thoroughly by pipetting.