Self-circularization of Linear DNA

 

 

1. In a microcentrifuge tube prepare a solution of linear DNA (25-50ng) in deionized water or TE buffer (10-35µl).
2. Add:
   • 10X ligation buffer 5µl,
   • 50% PEG 4000 solution (for blunt ends only)  5µl,
   • deionized water to 50µl,
   • T4 DNA Ligase 5u.
     Vortex the tube and spin down in a microcentrifuge for 3-5 seconds.
3. Incubate the mixture for 1 hour at 22°C .
4. Inactivate T4 DNA Ligase by heating reaction mixture at 65°C for 10 minutes.
5. Resulting reaction mixture can be used directly for transformation.

References

1. Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.
2. Current Protocols in Molecular Biology, vol. 1 (Ausubel, F.M., et al., ed.), John Wiley & Sons, Inc., Brooklyn, New York, 1994-2001.