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White and green screening with circular polymerase extension cloning for easy and reliable cloning
Author(s) -
Speltz Elizabeth B.,
Regan Lynne
Publication year - 2013
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.2268
Subject(s) - cloning (programming) , computational biology , protein engineering , biology , computer science , extension (predicate logic) , genetics , enzyme , programming language , biochemistry
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.