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Linker Ligation
(with T4 DNA Ligase)

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

   Linear DNA	5-7 µl (100-500 ng)
   Phosphorylated linkers	5 µl (1-2 µg)
   10X ligation buffer for T4 DNA Ligase	2 µl
   50% PEG 4000 solution	2 µl
   Water, nuclease-free	to 19.6 µl
   T4 DNA Ligase	0.4 µl (2 u)

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

2. Incubate the mixture for 1 hour at 22°C .
3. The resulting ligation products can be digested directly with restriction enzymes.

Note
Some restriction enzymes are susceptible to star activity in ligation buffer. Therefore, DNA should be precipitated with isopropanol or ethanol after ligation and dissolved in an appropriate buffer.

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