Journal: Nucleic Acids Research
Article Title: ExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes
Figure Lengend Snippet: Concerted action of in vitro assembly and full length RecE/RecT improves the efficiency of direct cloning. ( A ) A schematic diagram illustrating direct cloning of the 14-kb lux gene cluster from Photobacterium phosphoreum ANT-2200. The linear p15A-cm vector and target genomic segment have identical sequences at both ends. ( B ) Longer homology arms increase the cloning efficiency of ExoCET. The linear vector flanked by 25-, 40- or 80-bp homology arms was mixed with genomic DNA and treated with 0.02 U μl −1 T4pol at 25°C for 20 min before annealing and electroporation into arabinose induced Escherichia coli GB05-dir. Error bars, s.d.; n = 3. ( C ) Titration of T4pol amount for ExoCET. The linear vector with 80-bp homology arms and genomic DNA were treated as in (B) except the amount of T4pol was altered as indicated. ( D ) Incubation time of T4pol on cloning efficiency. As for (C) using 0.02 U μl −1 T4pol except the incubation time was altered as indicated. ( E ) Higher copy number of ETgA increases ExoCET cloning efficiency. As for (D) using 1 h and electroporation into arabinose induced E. coli GB05-dir (one copy of ETgA on the chromosome), GB2005 harboring pSC101-BAD-ETgA-tet (approximately five copies of ETgA on pSC101 plasmids) or GB05-dir harboring pSC101-BAD-ETgA-tet (approximately six copies of ETgA ) as indicated. ( F ) ExoCET increases direct cloning efficiency. As for (E) using E. coli GB05-dir harboring pSC101-BAD-ETgA-tet (ExoCET) or omission of T4pol from the in vitro assembly (ETgA) or omission of the arabinose induction of pSC101-BAD-ETgA-tet (T4pol). ( G ) As for (F) except the 53 kb plu2670 gene cluster was directly cloned. Accuracy denotes the success of direct cloning as evaluated by restriction digestions ( Supplementary Figure S4 ). Each experiment was performed in triplicate ( n = 3) and error bars show standard deviation (s.d).
Article Snippet: Assembly reactions with other exonucleases were cycled as follows: T5 exonuclease (T5exo; NEB, cat. no. M0363): 50°C for 30 min, then held at 4°C; T7 exonuclease (T7exo; NEB, cat. no. M0263): 25°C for 20 min, 50°C for 30 min, then held at 4°C; DNA polymerase I Klenow fragment (Kle; NEB, cat. no. M0210), T7 DNA polymerase (T7pol; NEB, cat. no. M0274) and λ exonuclease (λexo; NEB, cat. no. M0262): 25°C for 20 min, 75°C for 20 min, 50°C for 30 min, then held at 4°C; Exonuclease III (ExoIII; NEB, cat. no. M0206): 37°C for 20 min, 75°C for 20 min, 50°C for 30 min, then held at 4°C; Phusion DNA polymerase (Phu; NEB, cat. no. M0530): 37°C for 20 min, 50°C for 30 min, then held at 4°C.
Techniques: In Vitro, Clone Assay, Plasmid Preparation, Electroporation, Titration, Incubation, Standard Deviation