Chapter title |
Simple Cloning and DNA Assembly in Escherichia coli by Prolonged Overlap Extension PCR
|
---|---|
Chapter number | 13 |
Book title |
DNA Cloning and Assembly Methods
|
Published in |
Methods in molecular biology, January 2014
|
DOI | 10.1007/978-1-62703-764-8_13 |
Pubmed ID | |
Book ISBNs |
978-1-62703-763-1, 978-1-62703-764-8
|
Authors |
Chun You, Y.-H. Percival Zhang |
Abstract |
We developed a simple method (Simple Cloning) for subcloning one, two, or three DNA fragments into any location of a targeted vector without the need for restriction enzyme, ligase, exonuclease, or recombinase. This cloning technology can be applied to a few common Escherichia coli hosts (e.g., BL21(DE3), DH5α, JM109, TOP10). The protocol includes three steps: (a) linear DNA fragments (i.e., the insert DNA and the vector backbone) with two overlap ends were generated by regular high-fidelity PCR, (b) the DNA multimers were generated based on these equimolar DNA templates by using prolonged overlap extension PCR (POE-PCR) without primers added, and (c) the POE-PCR product was transformed to E. coli strains directly. Because positive colony efficiencies are very high, it is not necessary to identify desired clones by using colony PCR. Simple Cloning provides a new cloning and DNA assembly method with great simplicity and flexibility. |
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