New England Biolabs, E7546S, NEBNext® Ultra™ II End Repair/dA-Tailing Module

This module is part of the Ultra Related Categories DNA Library Prep for Illumina,, RNA Library Prep for Illumina,, Next Generation Sequencing Library Preparation Specification Materials Required but not Supplied Magnetic rack or plate (e.g., NEBNext ® Magnetic Separation Rack (NEB #S1515S), Alpaqua ® 96S Super Magnet Plate (#A001322), or equivalent) FAQ Q: There are 3 NEBNext Ligation modules, the NEBNext Ultra II Ligation Module (#E7595), the NEBNext Ultra Ligation Module (#E7445) and the NEBNext Quick Ligation Module(#E6056). Which module should be used in conjuction with the NEBNext Ultra II End Repair/dA-Tailing Module? A: The NEBNext Ultra II End Repair/dA-Tailing Module should be used with the Ultra II workflow. This module is designed for use with the NEBNext Ultra II Ligation Module (#E7595 ) and is compatible with the Ultra II workflow. The NEBNext Ultra Ligation Module (#E7445) and the NEBNext Quick Ligation Module (#E6056). are not compatible with the Ultra II workflow. Q: How do I clean up the reaction once complete? A: The module is designed to go directly into the NEBNext Ultra II Ligation Module (#E7595), therefore there is no cleanup step required. For use with non-Ultra II workflows, use the following protocol for cleanup using SpriSelect or AMPure XP® Beads (Beckman Coulter, Inc.): For use with AMPure XP beads, allow the beads to warm to room temperature for at least 30 minutes prior to use. Vortex beads to resuspend. Add 108 μl (1.8X) of resuspended beads to the ligation reaction. Mix thoroughly on a vortex mixer or by pipetting up and down at least 10 times. Incubate for 5 minutes at room temperature. Put the tube/PCR plate on an appropriate magnetic stand to separate beads from supernatant. After the solution is clear (about 5 minutes), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain the DNA targets. Add 200 μl of 80% freshly prepared ethanol to the tube/PCR plate while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant. Repeat Step 6 once. Air dry beads for 5 minutes while the tube/PCR plate is on the magnetic stand with the lid open. Caution: Do not overdry the beads. This may result in lower recovery of DNA target. Remove the tube/plate from the magnet. Elute the DNA target from the beads by adding 47 μl of 10 mM Tris-HCl or 0.1X TE. Mix well on a vortex mixer or by pipetting up and down and incubate for 2 minutes at room temperature. Put the tube/PCR plate in the magnetic stand until the solution is clear.Without disturbing the bead pellet, carefully transfer 42 μl of the supernatant to a fresh, sterile microfuge tube Q: Can I store the reaction after it is finished in the thermocycler (ie when it reaches the 4°C hold)? A: If necessary, samples can be stored at -20°C; however, a slight loss in yield (~20%) may be observed. We recommend continuing with adaptor ligation or cleaning up the reaction before stopping. Q: What do I do if I see a precipitate in the Ultra II End Prep Reaction Buffer? A: It is not uncommon to see a white precipitation in the Ultra II End Repair Buffer. If this occurs, allow the mixture to come to room temperature and pipette the buffer several times to break up the precipitate, followed by a quick vortex to mix.