Linker Ligation
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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.
