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  • pbabb926
    Junior Member
    • Oct 2012
    • 3

    #1

    Biotin dNTP Mix in Illumina MatePair v2 kits

    Greetings all! Has anyone come across or made their own substitute for the Biotin dNTP mix in the Illumina MatePair v2 long-fragment DNA library kits?

    An older post discussed ideas for a DIY / homemade biotin-dNTP mix, but I don't think a consensus was reached, and the NEB biotin mix that was mentioned is now discontinued.
    http://seqanswers.com/forums/showthread.php?t=4608
    Last edited by pbabb926; 03-08-2013, 07:20 AM.
  • dded
    Junior Member
    • Mar 2012
    • 1

    #2
    illumina mate pair bootleg protocol

    We are likewise looking to bootleg the illumina mate pair library prep protocol, and would love hearing from anyone who has successfully done so.

    Based on the previous discussion referenced above, I am planning on the 15 minute end repair before spiking in biotinylated dATP at equal concentration to the dATP already present in the reaction. My thoughts here being that some lost product seems inevitable, either from not all fragments being labeled, or steric hindrance from multiple biotin molecules limiting binding/recovery. Since all library steps after Streptavidin capture are carried out on the beads I would prefer to have my loss on the lack of DNA molecules biotinylated rather than worry how the additional biotin molecules may be contorting the DNA for the rest of the protocol.

    Additionally, based on a NAR paper that came out last year (Nieuwerburgh, FV; et al; Nucleic Acids Research, 2012, e24), looking to use Plasmid-Safe ATP-Dependent DNase and Exonuclease I to digest non-circularized DNA.

    Finally, for circularization I see nothing to suggest T4 ligase is not the best choice so going with T4.

    This topic seems to have fallen out of favor with the last real discussion taking place back in 2010, but I would love any feedback anyone has the time to provide.

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