To return to the NEBNext Ultra II FS DNA Library Prep Kit product pages, just click here for NEB #E7805 (without beads) or NEB #E6177 (with beads).
|Low library yield||Over-fragmentation or under-fragmentation||Refer to the “Fragmentation” section below.|
|Low DNA quality||Sample must be purified DNA in an aqueous buffer; DNA in solutions containing enzyme-denaturing reagents will not work properly.|
|Improper size selection||Any DNA fragments above or below the target size will not be included in the library. Ensure DNA fragments are of the correct size. For optimal results, a fragmentation timecourse can help you select the optimal length of the fragmentation step for your samples; briefly, set up several identical reactions and compare the effects of a variety of fragmentation-step incubation times.|
|Adaptor is denatured||When diluting NEBNext adaptors, use 10 mM Tris HCl (pH 7.5-8.0) with 10 mM NaCl. Keep the adaptor on ice until use.|
|Insufficient mixing||Mix samples well by pipetting up and down; once you’ve added the final component, set your pipette to 80-90% of the total reaction volume and mix thoroughly. Keep the tip in the liquid to avoid the formation of bubbles. For enzymatic steps, follow the recommendation in the manual (usually 10 mix cycles). Try to avoid leaving sample in the pipette tip or on the source tube during transfer.|
|SPRI beads have dried before elution||Add Elution Buffer and mix before the beads turn lighter brown and start cracking. For additional tips about SPRI beads, view our video.|
|Incomplete ethanol removal during SPRI bead wash||Quickly spin the tube after the last ethanol wash at each SPRI bead step, place the tube on the magnet, and remove residual ethanol with a p10 tip. For additional tips about SPRI beads view our video.|
|SPRI bead sample loss||Mix slowly to minimize the number of droplets clinging to the inside of the tip, which may not recombine with the sample before the tip is ejected. Dispense the final bead-containing solution slowly into the sample tube so that the liquid stays together. Wait ~1 second before pushing the pipette to the second stop, allowing the total volume to be dispensed. When removing the supernatant, take care not to remove any beads. Check your tip over a white piece of paper. If beads are visible, dispense everything back into the tube and allow beads to resettle. For additional tips about SPRI beads view our video.|
|Incorrect fragment size||Starting DNA size too short||
If your input DNA is less than 200 bp (for example cfDNA), use Ultra II DNA without fragmentation (NEB #E7645).
For optimal results, a fragmentation timecourse can help you select the optimal length of the fragmentation step for your samples; briefly, set up several identical reactions and compare the effects of a variety of fragmentation-step incubation times.
|Fragmentation too fast||DNA containing salts and already-nicked DNA can fragment faster than expected. Clean up the DNA prior to library prep.|
|Fragmentation inhibited||Agents that slow or inhibit enzymatic activity can cause low or no fragmentation of the DNA. A cleanup step prior to library prep may help remove contaminants. The Ultra II FS DNA Library Prep Kit is designed for use with extracted and purified DNA.|
|Size Selection||The size-selection conditions provided in the manual are for samples in ligation mix. Samples in other buffers may require different SPRI bead ratios that should be experimentally determined.|
|Adaptor-dimer formation||Adaptor concentration too high||To remove excess adaptor-dimer, repeat the bead cleanup step using a 0.9x bead ratio. To prevent adaptor-dimer formation in subsequent libraries, optimize adaptor dilution based on your sample input, quality, and type using an adaptor titration experiment (e.g., compare the degree of adaptor-dimers in several identical reactions, varying only the amount of adaptor present). Adaptor titration may need to be repeated if the source of the sample changes (e.g., extraction method, tissue type, etc.).|
|Adaptor was pre-mixed with Ligation Master Mix||Do not add adaptor to ligation master mix. This can cause increased adaptor-dimer formation. For best results, add adaptor to sample, mix, and then add Ligase Master Mix and ligation enhancer. Mix again.|