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.