一、In a microcentrifuge tube prepare a solution of linear DNA (25-50ng) in deionized water or TE buffer (10-35µl).
二、Add:
（一）10X ligation buffer 5µl,
（二）50% PEG 4000 solution (for blunt ends only) 5µl,
（三）deionized water to 50µl,
（四）T4 5u.
Vortex the tube and spin down in a microcentrifuge for 3-5 seconds. DNA Ligase
三、Incubate the mixture for 1 hour at 22°C .
四、Inactivate T4 DNA Ligase by heating reaction mixture at 65°C for 10 minutes.
五、Resulting reaction mixture can be used directly for transformation.
References
Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.
Current Protocols in Molecular Biology, vol. 1 (Ausubel, F.M., et al., ed.), John Wiley & Sons, Inc., Brooklyn, New York, 1994-2001.
