无缝克隆试剂盒知识

图1. 碧云天无缝克隆试剂盒的工作原理图。 DH5α菌株的基因型 HT115(DE3)超级感受态细胞

1. Restriction enzyme- and ligase-dependent cloning

传统的克隆策略，缺点很突出，耗试剂，有酶切限制，操作繁琐。

2. Xi cloning

原理：The Xi cloning technique relies on bacterial in vivo homologous recombination for the directional assembly of DNA fragments with identical sequences at their ends (Liang et al., 2005).

原理：Transformation of E. coli with the amplified DNA fragment and digested vector allows for directional assembly of the fragments via the endogenous DNA repair machinery.
缺点：普通的感受态DH5α是缺少了重组酶的。
文献：Liang, X., Teng, A., Chen, S., Xia, D., Felgner, P.L., 2005. Rapid and enzymeless cloning of nucleic acid fragmetnss. United States Patent no. 6,936,470 B2.

3. DpnI-expressing E. coli BUNDpnI

Here, based on in vivo circularization of PCR products via homologous recombination and DpnI digestion of the parental template in E. coli, we describe a simple and rapid strategy to introduce deletions, insertions, and point mutations into any amplifiable site of the target genes as required.
操作步骤：

• The first step is PCR amplification.
• The second step is transformation of the PCR products into E. coli

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核心：为了使DpnI能够消化大肠杆菌中的原始模板，构建了DNA甲基化酶（DNA adenine methylase）缺陷的DpnI表达菌株
优势： As DpnI was expressed in vivo, there is no need to purchase the enzyme and no additional manipulation of PCR products is required; thus, mutagenesis is carried out in a cost-effective and time-efficient manner.
缺点：导入的菌株必须是这里给出的改造后的菌株 E. coli BUNDpnI，没有商业化。
文献：Site-directed mutagenesis by combination of homologous recombination and DpnI digestion of the plasmid template in Escherichia coli

4. 穿梭载体克隆法

扩增的片段和切开的载体直接导入到酵母中，利用酵母的同源重组组合成环状质粒，然后从酵母中提取质粒，再导入到DH5。这个载体是穿梭载体，可以在酵母和大肠杆菌之间穿梭。
缺点：多此一举，操作反而复杂，不如直接进行无缝克隆。
文献：Agrobacterium tumefaciens-mediated transformation: An efficient tool for insertional mutagenesis and targeted gene disruption in Harpophora oryzae

5. in vivo cloning

Briefly, pJC005.em was linearized by either PCR (primer pair oJC218-oJC219) followed by DpnI digest of template DNA or direct restriction digest using BtgZI. Linearized backbone (50 ng) and the SOE PCR product obtained as outlined above were transformed into chemically competent E. coli DB10 cells(也可以用DH5α). Colonies were checked 24 h later for the correct insert by colony PCR.

原理：The recombination-impaired E. coli strains nevertheless retains sufficient recombinase activity for in vivo assembly of DNA fragments with homologous ends
Cell extracts of these recA-negative strains can be used for in vitro recombination of DNA fragments with homologous ends before transformation into competent cells (SLiCE) [15], indicating that commonly used recA-negative E. coli cells contain all the necessary enzymatic activities for homologous DNA recombination

M5 HiPer DB10B Competent Cell（DB10感受态细胞）

文献：
In vivo cloning of up to 16 kb plasmids in E. coli is as simple as PCR.
Rapid, Efficient, and Cost-Effective Gene Editing of Enterococcus faecium with CRISPR-Cas12a

USER friendly cloning technique

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新型体外重组克隆方法