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  • Non-specific bands when using Illumina adapters?

    Hi all,
    I am new to the Illumina system. When I did the PCR and ran the gel using normal sets of primers. Everything is nice and clean. However, when I used the primers with Illumina adapters. I got non-specific bands and smear. Can someone help me please?
    PCR conditions are the same for both with/without the adapters.

    16S primers: 515'F/926r

    GTGBCAGCMGCCGCGGTAA
    CCGYCAATTYMTTTRAGTTT

    16S primers with adapters:

    5'[TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG]GTGBCAGCMGCCGCGGTAA-3'
    5’[GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG]CCGYCAATTYMTTTRAGTTT-3'

    16S PCR condition:

    95 C - 5 min
    35 cycles of 95 C - 30 sec/ 55.5 C - 30 sec / 72 C - 30 sec
    72 C - 10 min

    18S primers: Euk1391f/EukBr

    GTACACACCGCCCGTC
    TGATCCTTCTGCAGGTTCACCTAC

    with adapters:
    5’[TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG]GTACACACCGCCCGTC-3'
    5’[GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG]TGATCCTTCTGCAGGTTCACCTAC-3'

    18S PCR condition:

    94C - 3 min
    35 cycles of 94C - 45 sec/ 57C - 60 sec/ 72C - 90 sec
    72C - 10 min

    These are all environmental DNA which the original extracted DNA concentration are very low.

    Thank you!
    Kitty

  • #2
    Hi Kitty,

    This is typhical for Illumina primers witha adapters. This method works only for a highly good DNA template. I suggest you use the protocol Kircher&Meyer (https://www.ncbi.nlm.nih.gov/pubmed/20516186). For metagenomics we're using primers (V3 region of 16S rRNA):
    F: CCTACGGGRSGCAGCAG
    R: ACCGCGGCKGCTGGC
    Polymerase KAPA HiFi, reaction with addition of 3% DMSO (we work on ancient DNA, if you have modern DNA samples, DMSO supplement is unnecessary but you can try with it).
    PCR protocol:
    95C - 2min
    29 cycles (when we made more cycles we got bands in blak sample)
    98C - 20 sec
    54C - 20 sec
    72C - 15 sec
    72C - 5 min
    4C -

    Next stepas are in Kircher&Mayer protocol:

    Blut end by - NewEngland Quik Blunting Kit
    Adapter ligation - NewEngland Quik Ligation Kit
    Adapter fill-in - NewEngland Bst polymerase
    Indexing raction - KAPA HiFi

    If you have aditional question fell free.

    All the best
    Tomek

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