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las_vegas

金虫 (初入文坛)

[求助] 核酸外切酶I 使用 已有1人参与

核酸外切酶I作用在如下3’端突出的双链,

5'NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN3'
3'NNNNNNNNNNNNNNNNNNNN5'
<------------20个----------------><-----------16个---------->

能否分解突出的16个碱基,最后形成一个两端平滑,长度为20碱基对的双链么?

或者不发生作用?
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greeneese

铁杆木虫 (正式写手)

【答案】应助回帖

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感谢参与,应助指数 +1
las_vegas: 金币+10 2016-01-07 13:05:24
NEB公司关于E. Coli外切酶I的说明:

https://www.neb.com/faqs/1/01/01 ... e-used-to-blunt-dna


FAQ: Can Exonuclease I be used to blunt DNA?
No. Short ends are not a substrate. Use DNA Polymerase I, Large (Klenow) Fragment (NEB# M0210) to fill in 5' overhangs (also called 3' recessed ends) and chew back 3' overhangs or use Mung Bean Nuclease (NEB# M0250) to chew back 3' or 5' overhangs. Note: 3' overhangs can not be filled in.

NEB明确给出了否定回答,建议用绿豆核酸酶来做这事,或者用E.Coli聚合酶Klenow片段,通过聚合把粘性末端消除掉。
2楼2016-01-07 11:19:27
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