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What are the most critical steps in the library generation?

First Strand cDNA Synthesis:

  • Do not cool the mastermix containing E1 (step 3).

  • Have the RNA / Custom Targeted Primer or RNA / FS/ DTT / dT samples at 42 – 50 °C when adding the E1-containing mastermix (step 4) to avoid mishybridization.

  • Mix properly by pipetting.

  • Do not forget to shortly spin down the samples at room temperature before and after adding the E1-containing mastermix.

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