Linker Ligation
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,
deionized water to 20µl,
T4 DNA Ligase 2u.
Vortex the tube and spin down in a microcentrifuge for 3-5 seconds.Incubate the mixture for 1 hour at 22°C .
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.
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