Tagmentation temperature protocol for DNA Illumina prep PCR free

[anly]

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

[Ben3]

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.