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Simple Cloning and DNA Assembly in Escherichia coli by Prolonged Overlap Extension PCR

  • Chun You
  • Y.-H. Percival Zhang
Part of the Methods in Molecular Biology book series (MIMB, volume 1116)

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.

Key words

Enzyme-free cloning Escherichia coli DNA assembly Prolonged overlap extension PCR Simple Cloning Subcloning 

Notes

Acknowledgments

This work was supported mainly by the Shell GameChanger Program, DOE BioEnergy Science Center, DOE ARPA-E Petro, and the Virginia Tech CALS Biodesign, and NSF SBIR I grant to PZ.

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Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Chun You
    • 1
  • Y.-H. Percival Zhang
    • 1
    • 2
    • 3
  1. 1.Biological Systems Engineering DepartmentVirginia TechBlacksburgUSA
  2. 2.Institute for Critical Technology and Applied Science (ICTAS), Virginia TechBlacksburgUSA
  3. 3.Gate Fuels Inc.BlacksburgUSA

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