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.
- 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.
- Some restriction endonucleases are susceptible to star activity in ligation buffer. Therefore, DNA should be precipitated with isopropanol or ethanol after ligation and dissolved in an appropriate buffer.
Wu, R. et al., Meth. Enzymol., 152, 343, 1987.
Updated August 23, 2002 13:51