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Self-circularization of Linear DNA
(with T4 DNA Ligase)

1. In a microcentrifuge tube prepare the following reaction mixture:
   

Linear DNA	10-35 µl (25-50 ng)
10X ligation buffer for T4 DNA Ligase	5 µl
50% PEG 4000 solution (for blunt ends only)	5 µl
Water, nuclease-free	to 49 µl
T4 DNA Ligase	1 µl (5 u)

Vortex the tube and spin down in a microcentrifuge for 3-5 seconds.

1. Incubate the mixture for 1 hour at 22°C .
2. Resulting reaction mixture can be used directly for transformation.

Note

• DNA can be dissolved in nuclease-free water or TE buffer: 10 mM Tris-HCl, 1 mM EDTA (pH 7.8).
• An excess of ligation mixture with respect to competent cells may decrease the transformation efficiency. Prior to electro-transformation, remove T4 DNA Ligase from the ligation mixture by DNA extraction with chloroform. The extracted DNA can be further precipitated with ethanol.

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. Ausubel, F.M., et al., Current Protocols in Molecular Biology, vol. 1, John Wiley & Sons, Inc., Brooklyn, New York, 1994-2001.

Ordering Information

T4 DNA Ligase T4 DNA Ligase (with PEG) Rapid DNA Ligation Kit Rapid DNA Ligation & Transformation Kit CloneJET™ PCR Cloning Kit InsTAclone™ PCR Cloning Kit TransformAid™ Bacterial Transformation Kit Nb.Bpu10I T7 DNA Polymerase T4 DNA Polymerase Hot Start Taq DNA Polymerase TrueStart™ Taq DNA Polymerase Pfu DNA Polymerase Taq DNA Polymerase (native) Taq DNA Polymerase (recombinant) M-MuLV Reverse Transcriptase RevertAid™ M-MuLV Reverse Transcriptase RevertAid™ H Minus M-MuLV Reverse Transcriptase First Strand cDNA Synthesis Kit RevertAid™ First Strand cDNA Synthesis Kit RevertAid™ H Minus First Strand cDNA Synthesis Kit Calf Intestine Alkaline Phosphatase (CIAP) Shrimp Alkaline Phosphatase (SAP) 6X DNA Loading Dye & SDS Solution ATP Water, nuclease-free

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Updated sausio 31, 2008 16:27