Linker Ligation

Linker Ligation (with T4 DNA Ligase )

1. In a microcentrifuge tube prepare a solution of blunt ended, dephosphorylated DNA (100-500ng) in TE buffer (5-7µl).
2. Add 1-2µg of phosphorylated linkers in 5µl of TE buffer.
3. Add:
   • 10X ligation buffer 2µl,
   • 50% PEG 4000 solution   2µl,
   • deionized water to 20µl,
   • T4 DNA Ligase 2u.
     Vortex the tube and spin down in a microcentrifuge for 3-5 seconds.
4. Incubate the mixture for 1 hour at 22°C .
5. Inactivate T4 DNA Ligase by heating the reaction mixture at 65°C for 10 minutes. The resulting ligation products can be digested directly with restriction endonucleases.