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Protein Sci. 2013 Jun;22(6):859-64. doi: 10.1002/pro.2268. Epub 2013 May 8.

White and green screening with circular polymerase extension cloning for easy and reliable cloning.

Author information

1
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA.

Abstract

Cloning is an essential prerequisite to test protein design and engineering ideas. However, it is often time consuming, unreliable, and therefore frustrating. Here, we present a streamlined cloning strategy that incorporates a powerful white and green screening protocol to identify colonies with inserts. We use circular polymerase extension cloning, which is both ligation and sequence independent. Furthermore, our entire procedure requires only three quick steps and one enzyme making it easy to use, inexpensive, and tractable. We anticipate that this method will be particularly useful for protein engineers who frequently subclone or make focused deletion, insertion, or substitution libraries.

PMID:
23592493
PMCID:
PMC3690724
DOI:
10.1002/pro.2268
[Indexed for MEDLINE]
Free PMC Article

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