TITLE:
Selection Vector for Direct Cloning of Proof Reading Polymerase Chain Reaction Products Based on the Lethal ccdB Gene in Escherichia coli
AUTHORS:
Pascal Weibel, Miriam Ender, Jerzy Madon, Annelies S. Zinkernagel, Reto A. Schuepbach
KEYWORDS:
PCR-Cloning Vector; Blunt End; ccdB; Toxic Gene
JOURNAL NAME:
Advances in Microbiology,
Vol.3 No.1,
March
27,
2013
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 (0.2 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.