T4 DNA Ligase (5 U/µL)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Always use the supplied buffer to avoid high salt concentration or add ATP to a compatible buffer

Upstream tips
Always use the supplied buffer to avoid high salt concentration or add ATP to a compatible buffer

Publication protocol

Ligations of the linkers with 5′-OH ends
The ligations of linkers A–B, C–D, and E–F by using T4 DNA ligase were performed in 100 µl of T4 DNA ligase reaction mixture containing 1 x T4 DNA ligation buffer (40 mM Tris-HCl, 10 mM MgCl2, 10 mM DTT, and 0.5 mM ATP; pH 7.8 at 25°C), 1 µM of each oligo, and 0.25 Weiss units/µl of T4 DNA ligase (Fermentas, Lithuania; Promega, USA; and Takara, Japan). The ligations of linkers A–B, C–D, and E–F by using E. coli DNA ligase were performed in 50 µl of E. coli DNA ligase reaction mixture containing 1 x E. coli DNA ligation buffer (30 mM Tris-HCl, 4 mM MgCl2, 10 mM (NH4)2SO4, 1.2 mM EDTA, and 0.1 mM NAD+; pH 8.0 at 25°C), 1 x BSA (0.005%), 2 µM of each oligo, and 6 U/µl of E. coli DNA ligase (Takara). To figure out if the ligation of linkers A–B and E–F could be inhibited by (NH4)2SO4, the ligation of these linkers were performed in 100 µl of T4 DNA ligase reaction mixture containing 7.5 mM (NH4)2SO4. The other ligation conditions were the same as above. To see if T4 PNK could use NAD+ as its phosphate donor, oligos 2 and 3 of linkers A–B were separately preincubated with T4 PNK in 100 µl of E. coli DNA ligase reaction mixture containing 1 x E. coli DNA ligase buffer, 1 x BSA, 1 µM of each oligo, and 40 U of T4 PNK (Takara). This mixture without ATP was incubated at 37°C for 30 min, and then extracted once with an equal volume of fresh phenol/chloroform (1∶1), and once with chloroform/isopentyl alcohol (24∶1). DNA was precipitated with 3 volumes of 100% ethanol and 1/10th volume of 3 M sodium acetate (pH 5.2) at −20°C for 2 hrs. DNA pellets were washed twice with 300 µl of 75% cold ethanol, dried at room temperature for about 2 hrs, and resuspended in 4 µl of sterilized ddH2O. The ligation was then performed in 50 µl of E. coli DNA ligase reaction mixture containing the resuspended oligos 2 and 3 (4 µl each), 2 µM of each of oligos 1 and 4, and the other components mentioned above. All of the ligase reaction mixtures mentioned above and the negative controls without ligase were incubated at 18°C for 10 hrs.

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Manufacturer protocol

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