我是使用NEB的T4 DNA polymerase 照NEB网页提供的protocal做 (如下) http://www.neb.com/nebecomm/products/productM0203.asp Usage notes: Incubate at temperature suggested for specific protocol. Refer to specific protocol to determine recommended dNTP concentrations. T4 DNA Polymerase is active in any 1X NEB retriction enzyme reaction buffer and T4 DNA Ligase reaction buffer when supplemented with dNTPs. Application notes: Protocol for blunting ends by 3′ overhang removal and 3′ recessed end fill-in: DNA should be dissolved in any 1X restriction enzyme reaction NEBuffer or 1X T4 DNA Polymerase Reaction Buffer supplemented with 100 μM dNTPs. Add 1 unit T4 DNA Polymerase per microgram DNA and incubate 15 minutes at 12°C. Stop reaction by adding EDTA to a final concentration of 10 mM and heating to 75° C for 20 minutes (1,2). CAUTION: Elevated temperatures, excessive amounts of enzyme, failure to supplement with dNTPs or long reaction times may result in recessed ends due to the 3′→ 5′ exonuclease activity of the enzyme ------------------------------------------------------------------------------ T4 DNA polymerase可以让Sticky end平滑化(blunting) (有两个不同的sticky end想要用blunting方式) 之後想ligation起来(T4 DNA ligase) 可是并没有成功 想请问各位大大有谁用过的经验或撇步 提供给我参考看看吧 谢谢^^ --

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