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Tagmentation library prep is not working!



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  • Tagmentation library prep is not working!

    Hello all,

    I'm currently preparing library preps using a Tn5 enzyme. One day last week, library prep worked (although the fragment sizes were a bit too small). Since then, I've run it several times, and only one library has worked! I'm baffled by this. As far as I'm aware, I've been doing everything exactly the same. I wondered whether any experts might have a suggestion as to what is stopping it, or even find a hint as to what is going wrong in the TapeStation images below.

    I've been Ampure-purifying the DNA extracts to remove any potential inhibitors of the enzyme. Gels show that there is high molecular weight DNA present in the extracts. I am pipette-mixing the enzyme before use.

    I know that Tn5 is quite sensitive, so I was wondering whether the enzyme might have decayed due to freeze-thaw, having been transported under dry ice (which makes the solution solidify). However, the only real opportunity for this was about 5 minutes outside the -20*C freezer whilst I aliquoted it into Eppendorfs - seems like a narrow window, especially as I tried to keep the tubes on a cold block for most of that time.

    Is it at all possible from the traces below to infer whether the problem might lie in the tagmentation step, the PCR, the adaptor annealing, or something else? For instance, there are large peaks that appear to be at the length of the Tn5 adaptors: does this suggest that the adaptors are not charging properly to the Tn5 enzyme and are remaining in solution instead?

    I'm using Tn5ME-A, Tn5ME-B, and Tn5ME-REV adaptor oligos (33, 34, and 19 base pairs long, respectively). The i7 and i5 primers are 47 and 51 base pairs long.

    Many thanks if you have any suggestions!

    TapeStation image for when tagmentation worked (albeit not perfectly yet!):

    Library preps subsequently not working:

  • #2
    Could be you're not doing enough cycles of PCR. I'd suggest doing a qPCR on the post-tagmentation libraries. The ratio of input DNA to Tn5 dictates insert size and prep efficiency. Dilute the libraries and quant using the same PCR primers you're using to amp the libraries, in the presence of SYBR.