LinkerLigation

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.