Journal: Scientific reports

Article Title: IVA cloning: A single-tube universal cloning system exploiting bacterial In Vivo Assembly.

doi: 10.1038/srep27459

Figure Lengend Snippet: Figure 1. Method overview and primer design optimisation. (a) Schematic of the universal IVA cloning protocol consisting of a single PCR reaction, producing homologous linear ends, followed by DpnI digestion and transformation, where amplified DNA is assembled in vivo by recombination. Primer design is shown for each type of basic modification: insertion, deletions, site-directed mutagenesis and sub-cloning. For insertions, the new sequence is best included in Fw and Rv primers, acting as the homologous region (magenta). For deletions, the overlap can be incorporated in any one primer, homologous to the other primer (orange) with primers straddling the undesired region (grey). Mutagenesis is similarly performed, inversely amplifying outside the undesired codon (ATG), with the replacement encoded in the forward primer (TGC). (b) Sub- cloning involves the amplification of both vector and insert in a single tube with homologous regions to directionally control assembly (blue and yellow).

Article Snippet: Colonies were manually counted (number of colonies reported as Colony-Forming Units per plate (CFU/plate)) and successful plasmid construction was assessed by restriction digestion and/or Sanger sequencing (Beckman Coulter) of colony DNA.

Techniques: Cloning, Transformation Assay, Amplification, In Vivo, Modification, Mutagenesis, Subcloning, Sequencing, Plasmid Preparation, Control

Journal: Scientific reports

Article Title: IVA cloning: A single-tube universal cloning system exploiting bacterial In Vivo Assembly.

doi: 10.1038/srep27459

Figure Lengend Snippet: Figure 3. Basic molecular cloning procedures using IVA cloning. (a) Schematic depicting the simultaneous deletion of an IRES cassette (grey) and insertion of a linker sequence (yellow) in the GluA2-pIRES-EGFP vector. (b) Agarose gel showing the resulting amplification of insertions (I) and deletions (D). These include (Lane 2) insertion of a myc-tag at the N-terminus of GluA3 using phosphorylated primers (PP), and (Lane 3) IVA primers, (Lane 4) deletion of an N-terminal myc-tag in GluA2, (Lane 5) deletion of the N-terminal domain of GluA3 and (Lane 6) construction of a fusion GluA2-EGFP tandem construct by deleting the IRES cassette and introducing a linker. Number of colonies produced on transformation, and the percentage of colonies tested that contain the correct plasmid is shown below. MW = 1 kb DNA ladder. (c) Agarose gel of PCR products providing a comparison between IVA and QuikChange TM mutagenesis primers. An enhancement of the intensity is seen for IVA primers in all cases. Number of colonies and percentage of correct clones for IVA cloning are shown below. (d) Cycle-by-cycle comparison of the PCR product formation between IVA (■ green) and QuikChangeTM (● magenta) for the GluA4 G208C mutation over 24 cycles of PCR (normalised to maximum value as 100%, n = 3). The increased PCR yield of IVA is appreciable. (e) Agarose gel electrophoresis visualisation of PCR products for sub-cloning examples (GSG1L coding region into pIRES-mCherry and GluA2 coding region into pCDN4.1/TO) each showing two independent amplifications (Vector: V, Insert: I). Colony yields and percentage correct are shown below. (f) Alternative strategy for vectors not amenable to amplification, shown with the cloning of EGFP-Homer1c (Insert), subject to PCR, DpnI treatment and PCR purification, into the adeno-associated virus vector pAAV-CW3SL-EGFP (cut with NheI and XhoI, and gel purified. Agarose gel visualisation of vector post-digestion identifies gel purified fragment (V) alongside PCR amplified Insert (I).

Article Snippet: Colonies were manually counted (number of colonies reported as Colony-Forming Units per plate (CFU/plate)) and successful plasmid construction was assessed by restriction digestion and/or Sanger sequencing (Beckman Coulter) of colony DNA.

Techniques: Molecular Cloning, Cloning, Sequencing, Plasmid Preparation, Agarose Gel Electrophoresis, Amplification, Construct, Produced, Transformation Assay, Comparison, Mutagenesis, Clone Assay, Subcloning, Purification, Virus