Plasmid Construction Using GoldenGate and Hi-Fi Cloning

Plasmids were built using both GoldenGate and Hi-Fi cloning [113 (link), 114 (link)]. Sequences and annotations are provided in GenBank format on the Additional file 3. Briefly, DNA fragments were PCR-amplified using Phusion High-Fidelity DNA polymerase (Thermo Scientific). Primers used in PCR reactions for GoldenGate were designed to add BsaI sites in both extremities of the amplicon while the primers for HiFi cloning were designed to include a complementary overhang in their extremities [114 (link)]. Primer sequences and construction schemes for each plasmid are available from the corresponding authors, RPO and JTM, upon reasonable request. All PCR products were cloned into a modified pBluescriptII KS( +) (Addgene #62540) lacking BsaI restriction site in a one-step digestion/ligation using FastDigest SmaI or FastDigest EcoRV (Thermo Scientific) and T4 DNA-ligase (Invitrogen) with 10 mM ATP. Final plasmid assembly by GoldenGate cloning was conducted as described previously [114 (link)]. Successful cloning was confirmed by Sanger sequencing (GATC Biotech).

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Estevez-Castro C.F., Rodrigues M.F., Babarit A., Ferreira F.V., de Andrade E.G., Marois E., Cogni R., Aguiar E.R., Marques J.T, & Olmo R.P. (2024). Neofunctionalization driven by positive selection led to the retention of the loqs2 gene encoding an Aedes specific dsRNA binding protein. BMC Biology, 22, 14.

Publication 2024

Phusion High-Fidelity DNA polymerase pBluescriptII KS( +) FastDigest EcoRV T4 DNA-ligase

Corresponding Organization :

Other organizations : Universidade Federal de Minas Gerais, University of Oregon, Inserm, Université de Strasbourg, Centre National de la Recherche Scientifique, Universidade de São Paulo, Universidade Estadual de Santa Cruz

Top 5 similar protocols

Variable analysis

independent variables

GoldenGate cloning
Hi-Fi cloning

dependent variables

Successful cloning confirmed by Sanger sequencing

control variables

Modified pBluescriptII KS(+) (Addgene #62540) lacking BsaI restriction site
FastDigest SmaI or FastDigest EcoRV (Thermo Scientific) for one-step digestion/ligation
T4 DNA-ligase (Invitrogen) with 10 mM ATP for one-step digestion/ligation

controls

Positive control: Successful cloning confirmed by Sanger sequencing
Negative control: Not explicitly mentioned

Annotations

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