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【求助】3’末端切平

丁香园论坛

2052
请问有谁做过3’末端切平啊?我要做平末端连接,先将目的片段通过限制酶进行酶切,可两端产生的都是3'突出末端,不知道该用那种酶进行切平比较好,看见有好多都是补平的体系,现不知道具体用哪个公司的那种酶效果会比较好,是个新手,谢谢了!
neb不错,值得推荐。
若水无涯 您好!

您如果要做3‘端的切除,NEB有以下几种酶建议您可以考虑:
DNA Polymerase I, Large (Klenow) Fragment and T4 DNA Polymerase (NEB# M0203) are the best choices for chewing back 3' overhangs and filling in 5' overhangs (3' recessed ends).DNA Polymerase I, Large (Klenow) Fragment can be used at 25°C or room temperature, but T4 DNA Polymerase must be used at 12°C due to its robust exonuclease. Both work well in a wide variety of buffers. Vent DNA Polymerase (NEB# M0254) and Deep Vent DNA Polymerase (NEB# M0258) can also be used but ThermoPol buffer must be used, the reaction temperature is high, and the enzyme cannot be heat inactivated. Mung Bean Nuclease (NEB# M0250) will chew back 3' overhangs but the strong exonuclease activity combined with the lack of polymerase activity yield a lower percentage of blunt ends.

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