1x t4 ligase buffer  (New England Biolabs)

Journal: The Journal of Biological Chemistry

Article Title: Simple in vitro single-stranded linear and circular DNA preparation, functional selection, and validation using phosphor-derived modifications

doi: 10.1016/j.jbc.2025.110874

Figure Lengend Snippet: ssPCRP circularization using complementary strands. A , Schematic representation of ssPCRP circularization with 50, 42, and 35 complementary bases bridging oligonucleotide containing TT(±T)TT at the ligation site. B , ssPCRP circularization with T4 DNA ligase. Lane M: marker. Lanes 1, 3, and 5: circularization reaction in the presence of 50, 42, and 35 bp complementary strands (TTTT sequences at ligation sites) and lanes 2, 4, and 6 in combination with 50, 42, and 35 bp complementary strands with an additional T nucleotide at the ligation site (TTTTT) at a final concentration of 1 μM, respectively. Lane 7: ssPCRP treatment with exonuclease I. Lane 8: ssPCRP. C , ssPCRP head-to-tail sealing using Taq DNA ligase enzyme. The order of the lanes is the same as A . D , schematic illustration of ssPCRP circularization using 35 bases of complementary strand–containing specific sequences at the ligation site. E and F , ssPCRP circularization via T4 and Taq DNA ligase enzymes, respectively. Lane M: marker; Lanes 1 and 2: circularization reactions using bridging oligonucleotides containing TACT or TATCT (additional T) sequence at the ligation position, respectively. ssPCRP, single-stranded PCR product.

Article Snippet: Circular library preparation was performed in final T4 ligation mixture of 100 μl including 1X T4 DNA ligase buffer (NEB), 1 μM phosphorylated library, 1 μM 35 bp complementary strand, 20 U T4 DNA ligase enzyme (NEB), and milliQ water for 1 h at 16 °C, which was followed by enzyme inactivation for 20 min at 80 °C.

Techniques: Ligation, Marker, Concentration Assay, Sequencing