New England Biolabs, M6630S, NEBNext® FFPE DNA Repair Mix

For superior performance, the NEBNext FFPE DNA Repair v2 Module ( Related Categories FFPE DNA,, Next Generation Sequencing Library Preparation Specification Materials Required but not Supplied Library Construction Reagents (e.g., NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB #E7645)) 100% Ethanol Nuclease-free Water 10 mM Tris-HCl, pH 8.0 or 0.1X TE DNA LoBind Tubes (Eppendorf #022431021) AMPure® XP Beads (Beckman Coulter, Inc. #A63881) NEBNext Multiplex Oligos for Illumina Magnetic rack or plate (e.g., NEBNext® Magnetic Separation Rack (NEB #S1515S), Alpaqua ® 96S Super Magnet Plate (#A001322), or equivalent) PCR Machine FAQ Q: Will the FFPE DNA Repair Mix ligate my DNA fragments? A: The ligase present in the FFPE DNA Repair Mix is Taq DNA ligase (NEB #M0208). This ligase effectively seals nicks in DNA, but is not effective at ligating fragments at a mismatch or at ligating blunt ended DNA. In fact, we have never observed this enzyme to ligate blunt ended DNA. Q: Will treating my DNA with the FFPE DNA Repair Mix hurt my downsteam reaction? A: No. We have not seen the FFPE DNA Repair Mix to have a negative effect on most downstream applications. The repair mix will act only on damaged bases, nicks and gaps. However, in specialized applications, such as methylation detection in damaged samples, there is the chance that the patch of bases filled in during the nick translation function of repair could erase methylation signatures. This effect is more pronounced in low DNA input experiments. Q: Will the FFPE DNA Repair Mix blunt the ends of the DNA? A: No. The polymerase present in FFPE DNA Repair Mix, Bst DNA polymerase, will fill-in 5’ overhangs, but will not remove 3’ overhangs. Furthermore, this polymerase will add a dA to the filled-in 5’ overhang. Q: Does the FFPE DNA Repair Mix insert random nucleotides into the sequence that it repairs? A: No, the polymerase in the mix adds the complementary bases that correspond to the template stand. The repair mix will not introduce artifacts into the sequence. Q: Does the FFPE DNA Repair Mix remove covalent modifications from DNA bases, such as methylation, biotin or digoxigenin? A: No, this repair mix will not specifically remove covalent modifications in the DNA bases, however bases adjacent to damage sites may be replaced with unmodified bases during the repair process. Q: Does the FFPE DNA Repair Mix repair DNA-protein crosslinks? A: No, the repair mix does not repair DNA-protein crosslinks. Q: Does the FFPE DNA Repair Mix fix blocked 3’ ends? A: Yes, the repair mix will polish the 3' end to a hydroxyl if there is a block at the 3' end. Q: Can the FFPE DNA Repair Mix repair damage in both single and double stranded DNA? Or, does it require double stranded DNA as a template? A: The FFPE DNA Repair Mix is for use with double stranded DNA. Some of the DNA repair enzymes contained in the mix can recognize damage in ssDNA, but the polymerization and ligation steps require dsDNA. Q: If I had a DNA template with mutation sites (ie. 8-oxoguanine or deaminated cytosines) that are directly adjacent to each other on opposite strands would treatment with FFPE DNA Repair Mix cause a double strand nick/break? A: We have not tried testing the repair of adjacent mutation sites on opposite strands. In this scenario, if the two damaged bases are removed at the same time, then a double stranded break could occur resulting in fragmented DNA. If, on the other hand, one reaction is faster than the other, and they do not occur simultaneously, then the FFPE DNA Repair Mix should repair them. Q: What gap lengths can be repaired with the FFPE DNA Repair Mix? A: The exact gap length is not known. Based on our experience, it is between 5-10 nucleotides. Q: When working with fragmented DNA, will the ends be ligated together by the FFPE DNA Repair Mix Reaction Mix? A: No, the Taq DNA ligase in the mix will only ligate where there is a nick in dsDNA. Q: How are abasic sites repaired by the FFPE DNA Repair Mix Reaction Mix? A: For dsDNA, the repair mix recognizes the abasic site and cuts the DNA backbone. The polymerase replaces the missing base with the correct base and the ligase ligates the DNA. Q: Can I use the FFPE DNA Repair Mix to repair other types of low quality DNA other than FFPE DNA? A: Yes. The repair mix will repair many basic DNA damage that is commonly found in other type of DNA of low quality, such as UV damaged DNA. Q: What is the difference between the NEBNext FFPE DNA Repair v2 Module (NEB #E7360) and the NEBNext FFPE DNA Repair Mix (NEB #M6630)? A: The NEBNext FFPE DNA Repair v2 Module contains the same repair activities as the NEBNext FFPE DNA Repair Mix but has been optimized for higher repair efficiency, a more streamlined workflow with NEBNext library preparation, and a more convenient reaction buffer containing all required buffer components for not only efficient DNA repair but downstream end repair and dA-tailing.