Selection Vector for Direct Cloning of Proof Reading Polymerase Chain Reaction Products Based on the Lethal <i>ccdB</i>Gene in <i>Escherichia coli </i>

作     者：Pascal Weibel Miriam Ender Jerzy Madon Annelies S. Zinkernagel Reto A. Schuepbach 

作者机构：Clinic of Infectious Diseases and Hospital Hygiene University Hospital Zurich University of Zurich Zurich Switzerland Surgical Intensive Care Medicine University Hospital Zurich University of Zurich Zurich Switzerland 

出 版 物：《Advances in Microbiology》 (微生物学（英文）)

年 卷 期：2013年第3卷第1期

页      面：14-20页

学科分类：1002[医学-临床医学] 100214[医学-肿瘤学] 10[医学] 

主　　题：PCR-Cloning Vector Blunt End ccdB Toxic Gene 

摘      要：Introducing PCR products into plasmids vectors is key for molecular techniques. Ideally cloning vectors are easy to construct, modify and propagate, neither require advanced techniques nor special equipment or reagents and efficiently incorporate PCR products at close to zero empty vector background. We provide an easy to engineer self-made cloning vector, neither requiring sophisticated tools or techniques nor advanced cloning knowledge. Through recombination we obtained the pUC18ccdB vector, carrying the ccdB suicide gene within the pUC18 backbone. When SmaI cleaved (within the ccdB) vector was T4 ligated with small (0.2 kbp) and intermediate (1.3 to 2.2 kbp) blunt end PCR-products and transformed into E. coli, the amount of clones with incorporated PCR product was comparable to commercial PCR-cloning kits and at a close to zero PCR product negative background. In conclusion we present a simple, versatile and cheap approach to an efficient “home made PCR-cloning vector that allows integration of crude blunt end PCR products at close to zero background.