New England Biolabs, E2612S, NEBNext® Microbiome DNA Enrichment Kit

Related Categories Microbiome DNA Enrichment,, Next Generation Sequencing Library Preparation Specification Materials Required but not Supplied λ DNA-HindIII Digest (NEB #N3012) 6-Tube Magnetic Separation Rack (NEB #S1506) Gel Loading Dye Blue (6X) (NEB #B7021) Nuclease-free water 0.8% Agarose Gel 1X TE Buffer, pH 7.5 Agencourt® AMPure® XP Beads (Beckman Coulter, Inc. #A63881) or Beckman SPRIselect Reagent Kit (Beckman Coulter, Inc. #B23317) 100% Ethanol for Alcohol Precipitation, 80% Ethanol for AMPure XP or SPRIselect DNA Low Bind Microcentrifuge Tubes Rotating Mixer Gel running equipment Heat block or Thermomixer Microcentrifuge Proteinase K, Molecular Biology Grade (optional; required for eluting captured host DNA) (NEB #P8107) FAQ Q: Can I incubate the DNA input sample with the MBD2-Fc-bound beads for a longer period of time? A: Yes, longer incubations are fine, as long as they do not exceed 4 hours. Q: How important is the MBD2-Fc bead to DNA ratio? A: It is vital that the ratio of MBD2-Fc-bound beads to DNA input used is 1 μl of MBD-Fc-bound beads per 6.25 ng DNA (or 160 μl MBD-Fc-bound beads per 1 μg DNA input). It is permissible to increase the amount of beads relative to DNA input, e.g. 320 μl of MBD2-Fc-bound magnetic beads for 2 μg DNA input. Q: What is the maximum volume of input DNA that I can use per reaction? A: We recommend up to 200 μl volume of sample (1 μg input DNA) for 160 μl Protein A-MBD2Fc beads. Higher sample volumes may be used for 160 μl Protein A-MBD2Fc beads, but we did not test the enrichment efficiency for volumes higher than 200 μl sample. Q: Will the procedure work on degraded DNA? A: We have observed that DNA size and quality does affect the microbiome DNA separation, as does the overall quantity of CpG methylated dinucleotides. For example, normal human fibroblast DNA (IMR-90) is methylated at 4–5% of the genome and is effectively separated by this technique (90% of human DNA is removed). Cancer DNA (HeLa, Jurkat) is less methylated globally at 3%, and enrichment is less efficient. Likewise, if the DNA fragment size is less than 15 Kb, enrichment will be less efficient. The larger and more CpG methylated the host DNA, the better the separation will be. Q: What is the best method for purifying the DNA after the enrichment? A: We recommend Ampure bead clean-up or Ethanol precipitation. Q: If my sample DNA is particularly degraded, as is the case with fecal microbiome samples, are there any protocol modifications that will enhance the performance of the NEBNext Microbiome DNA Enrichment Kit with these samples? A: While the NEBNext Microbiome DNA Enrichment Kit was designed for high-quality, intact DNA, users report successfully modifying the protocol to better repair low-quality DNA samples. Recommendations for how to adjust the protocol can be found in Chiou and Bergey 2018, Scientific Reports (2018) 8:1975 DOI:10.1038/s41598-018-20427-9; they include: Extra washes. Adjusting the ratio of beads/ host DNA based on qPCR results. 2 rounds of enrichment separated by an NaCl elution Please also see a protocol here: https://www.protocols.io/view/microbiome-dna-enrichment-for-fecal-seq-using-the-kqdg3xn21g25/v1