New England Biolabs, E3340S, NEBNext ULTRAEXPRESS® FS DNA Library Prep Kit

The NEBNext UltraExpress Related Categories Library Preparation for Illumina,, DNA Library Prep for Illumina,, Next Generation Sequencing Library Preparation FAQ Q: When preparing samples using the NEBNext UltraExpress® 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 TE. DNA can also be in 10 mM Tris pH 7.0-8.0 or H20. Q: What if I see a precipitate in the NEBNext UltraExpress® FS Reaction Buffer? A: It is not uncommon to see a white precipitate in the NEBNext UltraExpress® FS Buffer. If this occurs, allow the mixture to come to room temperature and pipette the buffer several times to resuspend the precipitate, followed by a quick vortex to mix. Q: Do I really need to vortex the NEBNext UltraExpress® FS Enzyme Mix? A: Yes, failure to vortex the NEBNext UltraExpress FS Enzyme Mix may produce variable results. 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: How much starting material must I use when preparing libraries with the NEBNext UltraExpress® FS DNA Library Prep Kit? A: This kit is suitable for 10 ng – 200 ng of intact input DNA. For input amounts outside of this range, end-user optimization will be necessary, and library quality may be affected. Q: What sample types can I use with the NEBNext UltraExpress® FS DNA Library Prep Kit? A: Compatible sample types include high-quality human, microbial, and plant genomic DNA and plasmid and amplicon DNA larger than 1 kb. We do not recommend this kit for use with FFPE DNA. For FFPE DNA, we recommend the NEBNext Ultrashear™ FFPE DNA Library Prep Kit (NEB #E6655). Q: Can I use the NEBNext UltraExpress® FS DNA Library Prep Kit for bisulfite conversion and EM-seq™ workflows? A: No, the NEBNext UltraExpress® FS kit is incompatible with NEBNext Enzymatic Methyl-seq (EM-seq; NEB #E7120) or bisulfite conversion workflows. We recommend you use NEBNext UltraShear™ (NEB #M7634). Q: Is it possible to use sheared DNA as input material with the NEBNext UltraExpress® FS DNA Library Prep Kit? A: For sheared DNA inputs, we recommend using the NEBNext UltraExpress® DNA Library Prep Kit (NEB #E3325) or NEBNext® Ultra™ II DNA Library Prep Kit (NEB #E7645). NEBNext UltraExpress® DNA Library Prep Kit (NEB #E3325) accommodates inputs of 10 ng – 200 ng of mechanically sheared DNA with a library prep workflow very similar to NEBNext UltraExpress® FS DNA. NEBNext® Ultra™ II DNA Library Prep Kit (NEB #E7645) accommodates inputs of 500 pg – 1 µg with a longer workflow time and customized conditions based on DNA input amount. Q: What is the difference between the NEBNext UltraExpress® FS DNA Library Prep Kit and the NEBNext® Ultra™ II FS DNA Library Prep Kit? How do I choose the right one? A: NEBNext UltraExpress® FS DNA Library Prep Kit is optimized for streamlined library prep with one adaptor concentration and PCR cycle number used for samples within the recommended input range (10 ng – 200 ng of intact DNA). NEBNext® Ultra™ II FS DNA Library Prep Kit has been extensively optimized to produce higher library yields at the highest quality. The Ultra II FS DNA Library Prep Kit has more customized conditions based on DNA input amount, more steps and cleanups, a longer workflow time, and a broader input range recommendation (0.1 ng – 500 ng of intact DNA). Both kits produce high yields of high-quality libraries, but we recommend choosing NEBNext UltraExpress® FS DNA for samples within the stated input amount range, where speed is paramount. We recommend choosing NEBNext Ultra II FS DNA for more complex and precious samples (e.g., low quality or low input). Q: Do the NEBNext UltraExpress® Library Prep kits come with beads? A: No, we recommend using the SPRIselect® Reagent Kit (Beckman Coulter, Inc. #B23317) or AMPure® XP Beads (Beckman Coulter, Inc. #A63881). Q: What should I do if I see adaptor dimer in my NEBNext UltraExpress® libraries? A: The phased cleanup step following PCR is specifically designed to reduce adaptor dimer; please follow the instructions for this step as written before attempting any additional recommendations. If you still see small amounts of adaptor dimer in samples, we recommend a 0.9X SPRI bead cleanup after pooling libraries or further optimization of the adaptor amount. Q: Can this kit be used with adaptors and primers from suppliers other 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®. Q: Can I use the NEBNext UltraExpress® FS DNA Library Prep Kit to prepare libraries for sequencing on Ion Torrent instruments? A: No, the adaptors required for Ion Torrent sequencing have blunt ends rather than 5´-T overhangs and are incompatible with the UltraExpress FS DNA workflow. Q: Does the fragmentation reagent in NEBNext UltraExpress® FS DNA kits contain NEBNext dsDNA Fragmentase®? A: No, the NEBNext UltraExpress® FS (Fragmentation System) DNA 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: Can the NEBNext UltraExpress® FS DNA Library Prep Kit provide sufficient library yields for target enrichment? A: Customized library PCR cycling conditions based on input amounts may be required for producing library yields of ~1 µg; see NEBNext UltraExpress® FS DNA Library Prep Kit manual appendices for recommended PCR cycling conditions.