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  • TruSeq DNA adapters in RNA-seq prep... concentration?

    Hi Everyone,

    I'm in a situation whereby I need to use two extra adapters than the 6 adapters that are currently shipped with each set of the TruSeq RNA-seq prep kit and at the moment we only have one set. We have both sets of adapters for the TruSeq Genomic DNA prep kit (i.e. all 12 adapters).

    I know the Genomic DNA and RNA-seq adapters are the same, but the concentration is different. Has anyone used the adapters from the DNA kit in the RNA sample prep? I think it used to be a 10 fold dilution of adapters using the 'old', non TruSeq kits.

    Cheers,

    Scott.

  • #2
    Can't you just check using absorbance at 260 nm (UV spec) if the UV spec or fluorescence (with oligreen) if UV spec is not sensitive enough?

    --
    Phillip

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    • #3
      Hi,

      Yes, I've measured them with picogreen... I guess I should have phrased the questions slightly differently...

      "Can anyone confirm that this will work, having actually switched the adapters after dilution and produced an experimentally tested library".

      I expect that it will be fine, but whenever I modify the protocols I always like to check whether anyone else has already tried it before I blow a few thousand dollars finding out the hard way :-)

      Cheers,

      Scott.

      Comment


      • #4
        I guess picogreen should not work well. It is a double stranded fluor that fluoresces very weakly in the presence of single stranded molecules. Hence it is not confounded much by RNA contamination of a DNA sample. For short oligonucleotides anneal over a short stretch I don't know you would get much signal.

        But if your concentration results were fairly linear over a set of dilutions, I guess you will be okay.

        Anyway, no, we have not tried it ourselves. Please let us know how it turns out.

        --
        Phillip

        Comment


        • #5
          Hi,

          Yes, I realise PG detects primarily dsDNA, but I think the adapters have sufficiently large double stranded regions to work with picogreen. We really only need a relative concentration rather than an abolute concentration.

          It appears that they're approximately 60 fold more dilute in the RNA-seq kit than in the genomic DNA kit.

          We diluted the DNA adapters 1 in 50 and the preps appear to have worked well.

          Cheers,

          Scott.

          Comment

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