Journal: PLoS ONE
Article Title: AQUA Cloning: A Versatile and Simple Enzyme-Free Cloning Approach
Figure Lengend Snippet: AQUA Cloning: a dvanced qu ick a ssembly cloning. (a) DNA parts are produced by PCR, or restriction digest (or both). Oligonucleotides are designed to contribute flanking homologous regions to adjacent DNA fragments of optimally 32 bp in length. DNA parts are assembled into a circular plasmid by sequence-determined directionality. (b) AQUA Cloning work-flow. (1) DNA parts are generated by PCR amplification, or derived from an enzymatic digestion. (2) Next, DNA parts are purified by gel-electrophoresis and (3) mixed and simply incubated in water prior to transformation into chemically competent E . coli Top10 cells for in vivo assembly. (4) Finally, obtained colonies are confirmed for correct assembly by standard methods such as analytical PCR, restriction digest, or comprehensive sequencing.
Article Snippet: E . coli strains The following commercially available strains of E . coli were also used for AQUA Cloning: One Shot TOP10 (Invitrogen cat. No. C4040-03, competency: > 109 CFU/μg), NEB5α (NEB, cat. No. C2987I, competency: 1–3 x 109 CFU/μg), NEB10β (NEB, cat. No. C3019I, competency: 1–3 x 109 CFU/μg), BL21 (DE3) (NEB, cat. No. C2527I, competency: 1–5 x 107 CFU/μg), JM109 (Promega, cat. No. L2005, competency: 108 CFU/μg).
Techniques: Clone Assay, Produced, Polymerase Chain Reaction, Plasmid Preparation, Sequencing, Flow Cytometry, Generated, Amplification, Derivative Assay, Purification, Nucleic Acid Electrophoresis, Incubation, Transformation Assay, In Vivo