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.
- 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
- Incubate the reaction mix for 30 minutes at 37°C.
- 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.
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