Optimal Cloning of PCR Fragments by Homologous Recombination in Escherichia coli
Fig 3
The test of an optimized recombinational cloning protocol.
(A) 0.5 ng of the pUC19 vector was amplified by PCR with the primers dest-f and dest-r. A sequence gap of 398 bp separates the 5′ termini of those primers. Five different templates were amplified with specific primers containing 20 bp long 5′ tails identical to the respective ends of the linear pUC19 vector. The S. japonicus region 6B was amplified by PCR with primers 6Bf and 6Br, while the cassettes zeoMX, natMX, and hphMX were produced with the primers MXf and MXr. Finally, the S. japonicus Ura4 gene was amplified with the primers Ura4f and Ura4r. The vector and PCR fragments were treated with DpnI and co-transformed in E. coli cells. Homologous recombination of each of these fragments with the pUC19 destination vector resulted in a different recombinant plasmid. (B) After transformation of each of the five constructs, 20 colonies were randomly picked to check for positive cloning events (+) by PCR with the primers pUC19f and pUC19r. The sequencing of one plasmid corresponding to each different construct confirmed a positive cloning event. Abbreviations are as described in Fig. 2.