New England Biolabs, E7103S, NEBNext® Ultra™ II DNA Library Prep with Sample Purification Beads

Related Categories DNA Library Prep for Illumina,, Analysis of DNA Methylation by Bisulfite Conversion,, Automation for NEBNext® NGS Library Prep, Specification Materials Required but not Supplied 80% Ethanol (freshly prepared) Nuclease-free Water 0.1X TE (1 mM Tris-HCl, pH 8.0, 0.1 mM EDTA) DNA LoBind Tubes (Eppendorf #022431021) NEBNext Multiplex Oligos for Illumina (for a full list, visit www.neb.com/oligos) Magnetic rack or plate (e.g., NEBNext® Magnetic Separation Rack (NEB #S1515S), Alpaqua ® 96S Super Magnet Plate (#A001322), or equivalent) PCR Machine Optional: 10 mM Tris-HCl, pH 8.0 or Tris/NaCl, pH 8.0 (10 mM Tris, 10 mM NaCl) (for adaptor dilution of DNA input ≤ 100 ng) Storage Temperature: Package 1: NEBNext Ultra II DNA Library Prep Kit for Illumina - Store at -20°C Package 2: NEBNext Sample Purification Beads - Store at Room Temperature. Do not freeze. FAQ Q: For what type of sample prep/library construction can I use the NEBNext Ultra II DNA Library Prep kit for Illumina? A: The NEBNext Ultra II DNA Library Prep kit for Illumina is compatible with protocols requiring a 3´ A overhang on the library molecules to facilitate ligation to adapters containing a 5´ T overhang, such as library prep for sequencing on the Illumina platform. At this time we have validated the Ultra II DNA Library Prep Kit only on the Illumina platform. Q: What type and how much starting material do I need to use when preparing libraries using the NEBNext Ultra II DNA Library Prep Kit for Illumina? A: 500 pg -1 ug of sheared DNA is recommended. Q: Is size selection required and what is the recommended method? A: Yes, we do recommend size selection using SPRIselect or AMPure XP beads when making libraries from DNA inputs > 50 ng. A size selection is not recommended if DNA input is less than or equal to 50 ng. The Ultra II kit is also compatible with other size selection methods, including agarose gel and E-gel. A detailed size selection protocol is included, with the DNA fragment size ranging from 150bp to 500-700bp (final library size 250-800bp). Q: Can I use the NEBNext Ultra II DNA Library Prep Kit for Illumina reagents for preparing libraries for sequencing on the Ion Torrent PGM or Proton? A: No. The adapters provided for ThermoFisher’s Ion Torrent have blunt ends rather than 5´-T overhangs. Q: Can NEB provide any of the reagents for the cluster generation step or the actual sequencing run? A: No. Q: Are these reagents available in a customized format, larger format or 96 well formats? A: Yes, customized formats can be accommodated. Please contact our OEM department at custom@neb.com for more details. Q: I am currently using Agencourt Ampure XP beads. Will the NEBNext Sample Purification Beads be a suitable replacement? A: Yes, the NEBNext Sample Purification beads can replace Ampure beads in your workflow. Please follow the size selection and reaction cleanup protocols in the Ultra II DNA Library Prep Kit manual. Q: Can I use this NEBNext kit with adaptors and primers from other vendors than NEB? A: Yes, it should be possible to use adaptors and primers from vendors other than NEB with this NEBNext kit. The adaptor must be a T overhang adaptor and the final library yield may be slightly lower than when using the NEBNext adaptors for Illumina®. Instructions for use with non-NEBNext, full length barcoded adaptors and P5 and P7 universal PCR primers can be found in the manuals for the NEBNext indexed/ UMI adaptor manuals. These manuals can be found at neb.com/E7416 (RNA) or neb.com/E7395 (DNA). Choose the kit manual for the NEBNext kit you will be using. The manuals are written specifically for the NEBNext indexed/ UMI adaptors, the same protocol should work with other adaptors of similar design. We recommend performing a proof of principle experiment with the desired library prep kit and desired indexed adaptors and using expendable samples for optimization of experimental conditions. 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. Q: Does the kit include adaptor and primers? A: No. The kit can be used with the NEBNext Multiplex Oligos for Illumina. For a complete list, see NEBNext® Multiplex Oligos Selection Chart. For use with NEBNext library prep kits, consult the library prep kit manual. The multiplex oligo kit specific manuals also contain tips for setting up PCR reactions. For color balancing options, please see NEBNext® Index Oligo Selector for valid barcode combinations.