User:Reef Koh Junjie/Notebook/DNA insert ligation into vector DNA

Insert non-formatted text hereIn 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. Incubate the mixture for 1 hour at 22°C. 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 Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001. Ausubel, F.M., et al., Current Protocols in Molecular Biology, vol. 1, John Wiley & Sons, Inc., Brooklyn, New York, 1994-2001.