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DNA Insert Ligation into Vector DNA
(with T4 DNA Ligase)

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

   Linear vector DNA	5-10 µl (50-400 ng)
   Insert DNA	use a 1:1 up to a 3:1 molar ratio of insert DNA termini to vector DNA
   10X ligation buffer for T4 DNA Ligase	2 µl
   50% PEG 4000 solution (for blunt ends only)	2 µl
   Water, nuclease-free	to 20 µl
   T4 DNA Ligase	0.2-0.4 µl (1-2 u) for sticky ends 1 µl (5 u) for blunt ends

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

2. Incubate the mixture for 1 hour at 22°C.
3. Use the mixture for transformation.

Note

• If the yield of ligation product is insufficient, prolong the reaction time (overnight).
• 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 Shrimp Alkaline Phosphatase (SAP) 6X DNA Loading Dye & SDS Solution ATP Water, nuclease-free

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Updated birželio 30, 2008 12:40