New England Biolabs, E7430L, NEBNext® Ultra™ II FS DNA PCR-free Library Prep Kit for Illumina®

The NEBNext Ultra II FS DNA PCR-free Library Prep Kit for Illumina offers an amplification-free workflow for DNA-seq based on the streamlined and reliable Ultra II FS workflow. Starting with as little as 50 ng of DNA, get the high-quality, high-yield libraries that you need without PCR bias. Related Categories Validation of CRISPR-based Gene Editing,, DNA Library Prep for Illumina,, Automation for NEBNext® NGS Library Prep, FAQ Q: What is the main application of the NEBNext Ultra II FS DNA PCR-free Library Prep Kit? A: The NEBNext Ultra II FS DNA PCR-free Library Prep Kit is optimized for preparation of high quality, amplification-free libraries for whole genome sequencing on the Illumina platforms. Q: What additional reagents do I need to complete the Ultra II FS DNA PCR-free workflow? A: In addition to the NEBNext Ultra II FS DNA PCR-free Library Prep Kit, you will need the NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Set 1) (NEB #E7395) to complete the workflow. Please be aware that all other oligos from NEB are not compatible with the PCR-free workflow. Q: Why is DTT included in the Ultra II FS DNA PCR-free kit? A: Addition of DTT in the fragmentation/end prep step helps to achieve the long insert sizes required for PCR-free libraries. Q: Can degraded samples be used for Ultra II FS DNA PCR-free library prep? A: No, we do not recommend degraded samples as input material for the PCR-free workflow. Use of degraded, low quality DNA might result in significantly lower yields and loss of samples during the cleanup steps. Q: What type of and how much starting material do I need when preparing libraries with the NEBNext Ultra II FS DNA PCR-free Library Prep Kit? A: This kit is suitable for 100 ng – 0.5 µg of intact, high-quality DNA using the standard protocol. In addition, we provide a low-input protocol (50 -99 ng) to support users for whom obtaining sufficient sample for the standard protocol is not possible. Q: When preparing samples using the NEBNext Ultra II FS DNA PCR-free Library Prep Kit, can my input DNA be in an EDTA-containing solution? 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 H2O. 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: 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: Following the Fragmentation and End Prep steps of the NEBNext Ultra II FS PCR-free workflow, can the reactions be kept at 4˚C? A: No. We recommend moving immediately to the Adaptor Ligation step once the reaction temperature reaches 4˚C. Leaving the end-prepped DNA at 4˚C could result in lower library yields. 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: Following the Adaptor Ligation step, can the reactions be kept at 4˚C? A: No. We recommend moving immediately to bead clean-up steps once the reaction temperature reaches 4˚C. Q: Can I use the NEBNext Ultra II FS DNA PCR-free Library Prep Kit for bisulfite conversion or EM-seq™ workflows? A: No, the Ultra II FS DNA PCR-free kit is not compatible with bisulfite conversion or EM-seq workflows. We recommend using mechanical shearing and the NEBNext Ultra II Library Prep Kit (NEB #E7645) for this application. Q: Can I use the NEBNext Ultra II FS DNA PCR-free 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 3´-T overhangs and are not compatible with the Ultra II FS DNA PCR-free workflow. Q: How do I quantify my PCR-free libraries? A: Only qPCR-based methods (such as the NEBNext Library Quant Kit for Illumina (NEB # E7630)) can be used for accurate quantitation of PCR-free libraries. Other methods will quantify fragments that may not have adaptors at both ends, and therefore will not generate clusters or sequence. Q: Can I assess PCR-free library quality on a BioAnalyzer and/or TapeStation? A: Precautions should be taken when using Bioanalyzer and TapeStation to assess quality of PCR-free libraries. Bioanalyzer and TapeStation can be informative at revealing whether PCR-free libraries are contaminated with leftover adaptors or adaptor dimers. However, size estimation and library quantitation are not accurate on Bioanalyzer and TapeStation. The long, single stranded region of the PCR-free adaptors interfere with migration of PCR-free libraries, making them appear to be much larger. In addition, Bioanalzyer and TapeStation cannot differentiate between input DNA fragments and DNA fragments with one or two adaptors ligated, which makes them unsuitable for quantitation. We recommend qPCR for library quantitation of PCR-free libraries. Q: Can I use this NEBNext PCR-free kit with adaptors from vendors other than NEB? A: Yes, it is possible to use adaptors from vendors other than NEB with this NEBNext PCR-free library prep kit. The adaptors must be a T-overhang adaptor and contain all of the necessary sequences required for sequencing on Illumina platforms. Q: Does the PCR-free kit include adaptor and primers? A: No. The kit can be used with the NEBNext Unique Dual Index UMI Adaptors for Illumina. For a complete list of compatible oligos, see the rightmost column of NEBNext® Multiplex Oligos Selection Chart. The NEBNext Unique Dual Index UMI Adaptors manuals also contain tips for setting up ligation reactions. For use with NEBNext library prep kits, consult the library prep kit manual. For color balancing options, please see NEBNext® Index Oligo Selector for valid barcode combinations.