New England Biolabs, E6177S, NEBNext® Ultra™ II FS DNA Library Prep with Sample Purification Beads

Related Categories DNA Fragmentation & RNA Fragmentation,, DNA Library Prep for Illumina,, Automation for NEBNext® NGS Library Prep, Specification Materials Required but not Supplied 80% Ethanol (freshly prepared) Nuclease-free water 0.2 ml thin wall PCR tubes NEBNext Singleplex or Multiplex Oligos for Illumina (NEB #E7350, #E7335, #E7500, #E6609, #E7710, #E7730 or #E7600) Magnetic rack or plate (e.g., NEBNext® Magnetic Separation Rack (NEB #S1515S), Alpaqua ® 96S Super Magnet Plate (#A001322), or equivalent) PCR machine Vortex Microcentrifuge For NEB #E7805 only: SPRIselect® Reagent Kit (Beckman Coulter, Inc. #B23317) or AMPure® XP Beads (Beckman Coulter, Inc. #A63881) Optional: 10 mM Tris-HCl, pH 8.0 with 10 mM NaCl (for adaptor dilution of DNA input < 100 ng) FAQ Q: When preparing samples using the NEBNext Ultra II FS DNA Library Prep Kit, can my input DNA be in EDTA-containing solutions? A: Yes, we recommend that the DNA be in 1X TE or low EDTA-containing TE. DNA can also be in 10 mM Tris pH 7.0-8.0 or H20. Q: What do I do if I see a precipitate in the Ultra II FS Reaction Buffer? A: It is not uncommon to see precipitation in the Ultra II FS Reaction Buffer. If this occurs, pipette the buffer several times to break up the precipitate, followed by a quick vortex to mix. You may still see a small amount of precipitation, but this will not affect performance. Q: Following the Fragmentation and End Prep steps of the NEBNext Ultra II FS workflow can the reactions be kept at 4° C? A: Yes, the fragmented, end-prepped DNA can be kept at 4˚C for several hours without any appreciable effect. However, after overnight storage at 4˚C a decrease in yield of approximately 20% can be expected. Q: Do I really need to vortex the Ultra II FS Enzyme Mix? A: Yes, failure to vortex the Ultra II FS Enzyme Mix may produce variable results. Q: What type of and how much starting material do I need to use when preparing libraries with the NEBNext Ultra II FS DNA Library Prep Kit A: This kit is suitable for 100 pg – 0.5 µg of intact input DNA. Q: Can I use the NEBNext Ultra II FS DNA Library Prep Kit for bisulfite conversion and EMSeq workflows? A: No, the Ultra II FS kit is not compatible with bisulfite conversion or EMSeq workflows. We recommend you use mechanical shearing and the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645) for this application. Q: The ligation reaction is very viscous. What will happen if it is not mixed properly? A: If the ligation reaction is not mixed properly, low library yields may result. To ensure proper mixing, the reaction can be pipetted up and down 10 times, or vortexed for 3 seconds. Q: Can I use the NEBNext Ultra II FS DNA Library Prep Kit to prepare libraries for sequencing on Ion Torrent sequencing instruments? A: No, the adaptors required for Ion Torrent sequencing have blunt ends rather than 5´-T overhangs and are not compatible with the Ultra II FS workflow. Q: What is the concentration of my index primers? A: NEBNext index primers are 10 µM concentration. Q: I am currently using Agencourt AMPure® XP beads in my library prep workflows. Will the NEBNext Sample Purification Beads be a suitable replacement? A: Yes, the NEBNext Sample Purification beads are SPRIselect® beads from Beckman Coulter and can replace AMPure beads in your workflow. Please follow the size selection and reaction cleanup protocols in the Ultra II FS 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: Does the fragmentation reagent included in NEBNext Ultra® II FS DNA kits contain NEBNext dsDNA Fragmentase®? A: No, the Ultra II FS (Fragmentation System) products contain a unique mix of enzymes that fragment, end repair, and dA tail DNA, in a single tube. Fragmentase is not included in the FS enzyme mix. Q: Is the NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina® compatible with all sample types? A: The NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina® has been tested in many sequencing applications including whole genome sequencing, hybrid capture, amplicon sequencing, and copy number assessment, etc. It has been designed to maximize the number of molecules incorporated into a sequencing library. As a result, this method is able to produce diverse libraries even from very small amounts of input DNA (< 1 ng). Please keep in mind the following: Modifications present in the DNA input, such as methylation and hydroxymethylation, can be replaced by their canonical form when using FS. This method should not be used for applications where modified bases are being studied (e.g. EM-seq and bisulfite sequencing). At very low frequency (typically less than 0.1%), FS may introduce artifacts identified as chimeras or tandem reads in downstream analysis. These artifacts can be filtered using fgbio FindSwitchbackReads. For FFPE samples, we have observed increased noise in some FS libraries. However, due to the diversity of FFPE damage, it is not possible to predict which FFPE samples will exhibit higher noise. For best data quality, we recommend using the NEBNext Ultrashear FFPE DNA Library Prep Kit (NEB #E6655) or the NEBNext FFPE DNA Library Prep Kit (NEB #E6650). We encourage users to carefully evaluate any library prep method to ensure it is appropriate for the specific sample type being studied. Please reach out to info@neb.com if you have questions about your specific application. 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.