Partial?Endonuclease?Digestion

Prepare a 100 µl reaction mixture containing the DNA of interest in the appropriate 1X restriction enzyme buffer. Divide the reaction mixture between five prechilled microcentrifuge tubes such that tube 1 contains 30 µl, tubes 2 through 4 contain 20 µl, and tube 5 contains 10 µl (see diagram below). Place the tubes on ice.

Add restriction endonuclease (3-10 units of enzyme per µg of DNA ) to tube 1, mix quickly, and place the tube on ice.

Add 10 µl from tube 1 to tube 2, mix quickly, and place on ice. Continue serial dilutions using 10 µl per transfer. It is critically important to use a fresh pipette tip for each transfer.

Incubate the tubes for 15 minutes at the temperature appropriate for the restriction enzyme used.

Immediately bring the volume of all tubes to 100 µl by adding 80 µl sdH₂O. Quickly add 100 µl phenol, mix by vortexing, and separate phases by centrifuging 5 minutes. Recover the aqueous phase, transfer it to a new tube, and combine it with 100 µl chloroform:isoamyl alcohol (24:1). Mix, centrifuge 5 minutes. Transfer the aqueous phase to a new tube. Add 15 µl 3 M sodium acetate and 250 µl 95% ethanol. Precipitate the DNA overnight at -20℃. Recover the DNA by centrifugation for 15 minutes. Dry the pellet.

Resuspend the pellet in 17 µl sdH₂O, 2 µl 10x ligase buffer, and 1 µl T4 DNA ligase. Remove 2 µl from each tube to analyze the extent of digestion. Incubate the ligations overnight at 18℃. The ligation mixtures can be used directly to transform competent cells.

It is critically important that digestion time be controlled precisely. Therefore, work quickly, always keeping the tubes on ice to minimize digestion during dilution steps. It is also helpful to keep small the number of such digestions performed simultaneously.