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  • End Repair of cDNA Library

    For use with NEBNext® mRNA Library Prep Reagent Set for Illumina® (E6100) and NEBNext® mRNA Library Prep Master Mix Set for Illumina® (E6110).

    Protocol

    1. Mix the following components in a sterile 1.5 ml microcentrifuge tube:
      Purified double-stranded cDNA: 50 μl
      Nuclease-free Water: 25 μl
      10X Phosphorylation Reaction Buffer: 10 μl
      Deoxynucleotide Solution Mix: 4 μl
      T4 DNA Polymerase: 5 μl
      E. coli DNA Polymerase I, Large (Klenow) Fragment: 1 μl
      T4 Polynucleotide Kinase: 5 μl 
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      Total volume: 100 μl
    2. Incubate in a heat block for 30 minutes at 20°C.
    3. Purify end repaired DNA using a PCR column purification kit, purifying the sample on one column and eluting in 32 μl sterile water or elution buffer.