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Selection vector for direct cloning of proof reading polymerase chain reaction products based on the lethal ccdB gene in Escherichia Coli


Weibel, Pascal; Ender, Miriam; Madon, Jerzy; Zinkernagel, Annelies; Schuepbach, Reto (2013). Selection vector for direct cloning of proof reading polymerase chain reaction products based on the lethal ccdB gene in Escherichia Coli. Advances in Microbiology, 3(1):14-20.

Abstract

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 (0.2kbp) and intermediate (1.3 to 2.2kbp) 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.

Abstract

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 (0.2kbp) and intermediate (1.3 to 2.2kbp) 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.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > University Hospital Zurich > Division of Surgical Research
04 Faculty of Medicine > University Hospital Zurich > Institute of Intensive Care Medicine
04 Faculty of Medicine > University Hospital Zurich > Clinic for Infectious Diseases
Dewey Decimal Classification:610 Medicine & health
Language:English
Date:2013
Deposited On:06 Nov 2013 13:45
Last Modified:05 Apr 2016 17:07
Publisher:Scientific Research Publishing
ISSN:2165-3402
Funders:Swiss National Founda- tion grant# PZ00P3_136639
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.4236/aim.2013.31002

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