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  • Tagmentation temperature protocol for DNA Illumina prep PCR free

    Hi,

    We are testing running Illumina DNA prep PCR free and we noticed that our colleague had used the wrong protocol for ELM, instead of 37°C for 5 minutes followed by 50°C for 5 minutes, the following protocol was used: 37°C for 5 minutes followed by 50°C for 30 sec, meaning a 4.5 min shorter run at 50°C. Does anyone know what this step does and how it can impact performance.

    We are also interested in any aspects that can improve data, in particular GC rich dropout regions.

    /Anna

  • #2
    Hi Anna,

    The ELM is short for Extension Ligation Mix and it helps ligate the adapters. I'm believe that during the 37°C for 5 minutes, the adapters are being ligated and then during the 50°C section, the ligases are being inactivated.

    My assumption is that the libraries will turn out mostly fine if you did a thorough cleanup after the ligation step. You just may have more adapter dimers. At the end make sure you check your libraries on a BioAnalyzer or TapeStation to make sure they are the correct length.

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