T4 DNA 连接酶缓冲液

dATP是一个竞争性抑制剂。磷酸基团将降低连接效率。连接缓冲液中的去垢剂可能不会影响酶活性。高浓度(0.2 M)的Na2+, K+, Cs+, Li+, 和NH4+几乎可以完全抑制酶活性。多胺、精胺和亚精胺也是抑制剂。

在一个连接反应中，至少有一个分子（例如插入片段或载体）必须是磷酸化的。连接反应取决于DNA分子上5’磷酸的存在。去磷酸化载体与限制性内切酶消化生成的插入片段（磷酸化的）结合，是最常见的方式。尽管DNA只有一条链可连接在接合点，但是也可形成稳定环状结构的分子，前提条件是插入片段足够长，双链杂交产生的力量能够维持分子的环状结构。

这取决于您的应用。对于含有黏性末端的双链DNA片段之间的连接，两种酶都能使用。大肠杆菌DNA连接酶需要β-NAD存在，而T4 DNA连接酶需要ATP。然而，只有T4 DNA连接酶可以连接平末端DNA片段；大肠杆菌连接酶不可用于这类片段的连接。

大肠杆菌DNA连接酶通常用于第二链cDNA合成中修复缺口。在第二链cDNA合成中，T4 DNA连接酶不可代替大肠杆菌DNA连接酶，因为T4 DNA连接酶对平末端ds cDNA片段连接的功能，可能导致形成嵌合插入。

dATP is a competitive inhibitor. Phosphate will reduce ligation efficiency. Detergents in your ligation buffer will likely not affect activity. High levels (0.2M) Na2+, K+, Cs+, Li+, and NH4+ inhibit the enzyme almost completely. Polyamines, spermine, and spermidine also serve as inhibitors.

At least one molecule in a ligase reaction (i.e., insert or vector) must be phosphorylated. Ligation reactions are dependent on the presence of a 5' phosphate on the DNA molecules. The ligation of a dephosphorylated vector with an insert generated from a restriction enzyme digest (phosphorylated) is most routinely performed. Although only one strand of the DNA ligates at a junction point, the molecule can form a stable circle, providing that the insert is large enough for hybridization to maintain the molecule in a circular form.