This step ligates a phosphorylated T-Adapter with a 3’-T overhang to the ends of the DNA fragments from primer extension 3’ L1H products that have 3’-A overhangs generated at Step 2. The reaction adds a specific adapter sequence to the 3’ ends of the genomic fragment.

  1. Prepare the reaction mix on ice in the following order:

    Component Volume
    Extended 3’ L1H products from Step 2 30 µl
    T4 DNA Ligase Buffer (10X) 5 µl
    T-Adapter 2 µl
    PCR-Grade Water 12.5 µl
    T4 DNA Ligase (100X) 0.5 µl
    Total 50 µl
  1. Incubate the reaction mix for 30 minutes at 37°C.
  1. Follow the instructions in the QIAquick PCR Purification Kit to purify the reaction on one QIAquick column, eluting in 30 μl of QIAGEN EB buffer.
Last modified: Mar 08, 2017

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