New England Biolabs, E3325S, NEBNext ULTRAEXPRESS® DNA Library Prep Kit

The NEBNext UltraExpress DNA Library Prep Kit is the latest generation of NEBNext DNA library prep, with a fast, streamlined workflow. In under 2 hours, the protocol enables the creation of high-yield, high-quality libraries, from a broad input amount range, while generating less plastic waste. Further, with simplicity at the forefront, the kit features a single protocol for all input amounts, making NEBNext UltraExpress DNA Library Prep well suited for all throughputs. Related Categories Library Preparation for Illumina,, DNA Library Prep for Illumina,, Next Generation Sequencing Library Preparation FAQ Q: What sample types can I use with the NEBNext UltraExpress® DNA Library Prep Kit? A: The NEBNext UltraExpressDNA Library Prep Kit is compatible with high-quality human genomic DNA, cell-free DNA (cfDNA) and DNA from other species (e.g., microbial, plant, etc.). We do not recommend this kit for use with FFPE DNA. For FFPE DNA, consider the NEBNext UltraShear FFPE DNA Library Prep Kit (NEB #E6655). Q: Is it possible to use enzymatic fragmentation upstream of NEBNext UltraExpress® DNA Library Prep Kit? A: Enzymatically fragmented samples may be used as input for this kit; however, this will require additional end-user optimization. Q: What is the recommended protocol for mechanical shearing upstream of NEBNext UltraExpress® DNA Library Prep? A: The NEBNext UltraExpress DNA Library Prep Kit has been tested for compatibility with the Covaris ML230 instrument with the following consumables and programs: 8 AFA-TUBE™ TPX Strip with caps (175bp program). Please refer to the manufacturer's instructions for other shearing protocols if you use a different instrument/model. Q: What is the difference between the NEBNext UltraExpress® DNA Library Prep Kit and the NEBNext® Ultra™ II DNA Library Prep Kit? How do I choose the right one? A: The NEBNext UltraExpress DNA Library Prep Kit has been optimized for streamlined library prep processing with one adaptor concentration and one PCR cycling condition used for all samples within the 10 – 200 ng input range. NEBNext Ultra II 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 (500 pg – 1 µg). Both kits produce high yields of high-quality libraries, but we recommend choosing NEBNext UltraExpress DNA for all of your standard samples within the stated input amount range, where speed is paramount. We recommend choosing NEBNext Ultra II DNA for more complex and precious samples (e.g., GC-rich, 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: What is the best way to quantify my NEBNext UltraExpress® DNA libraries? A: Libraries can be loaded on TapeStation using HSD1000 ScreenTape or a similar fragment analysis instrument for library quantification. However, for high-input DNA using the recommended PCR cycle number, there will be a second peak beyond the upper marker resulting from amplification in the non-linear amplification phase during late PCR cycles. Although the molecules generated during the non-linear amplification phase are sequencible to generate high-quality data, their different migration speed on ScreenTape causes inaccurate quantification of the library concentration. In this case, we recommend measuring library concentration on NanoDrop or Lunatic using 400 bp or the expected library size to convert the concentration values to nanomolar. For more information, please refer to the product manual. 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: 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.