SelfcircularizationofLinearDNA

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.