Linker Ligation (with T4 ) DNA Ligase

In a microcentrifuge tube prepare a solution of blunt ended, dephosphorylated DNA (100-500ng) in TE buffer (5-7μl).

Add 1-2μg of phosphorylated linkers in 5μl of TE buffer.

Add:

10X ligation buffer 2μl, 50% PEG 4000 solution 2μl, deionized water to 20μl, T4 2u.

Vortex the tube and spin down in a microcentrifuge for 3-5 seconds. DNA Ligase Incubate the mixture for 1 hour at 22℃ .

Inactivate T4 DNA Ligase by heating the reaction mixture at 65℃ for 10 minutes. The resulting ligation products can be digested directly with restriction endonucleases.