Quick Blunting™ 快速末端平齐化试剂盒将 DNA 不匹配的 5´ 或 3´ 突出末端转变为带有 5´ 磷酸化的平末端,以便有效地与平末端 DNA 克隆载体连接。用 T4 DNA 聚合酶(NEB #M0203)使 DNA 末端平齐化,该酶同时具有 3´→5´ 核酸外切酶活性和 5´→3´ 聚合酶活性。酶混合液中包含 T4 多...
5 min quick ligation
4d). Additionally, we developed an efficient protocol along with an improved buffer system (called TNT-Buffer; 50 mM Tris-HCl pH7.5, 2 mM DTT, 10 mM MgCl2, 1 mM ATP and 2% PPG) that allowed EarI and LguI enzymes to work well in combination with T4 DNA ligase in a “one...
Melting temperatures of oligonucleotides were determined using the New England Biolabs Tm Calculator (https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator). Four primers were used in egfp cloning experiment: EGFP-Fwd (5′- ATGGTGAGCAAGGGCGAG-3′, 18 bp), IntA-EGFP-Fwd (5...
Differently, the analogous reactions performed using the regular T4 DNA ligase buffer, the LR Reaction from the Gateway system or the isothermal (Gibson) assembly for cloning 1 element retrieved, respectively, 2.6 × 104 (SE ± 16.3%), 4.7 × 104 (SE ± ...