Transposition helper vector

Manufactured by Thermo Fisher Scientific

The Transposition helper vector is a plasmid designed to facilitate the genetic modification of target organisms through the process of transposition. It provides the necessary tools and components to enable the efficient integration of desired genetic elements into the genome of the host organism.

Automatically generated - may contain errors

Lab products found in correlation

Gentamycin, by Carl Roth (2 mentions) Insect xpress medium, by Lonza (2 mentions) Cellfectin reagent, by Thermo Fisher Scientific (2 mentions)

2 protocols using transposition helper vector

Recombinant Baculovirus Production in Sf9 Cells

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Escherichia coli DH10bac cells harboring the baculovirus genome (bacmid) and a transposition helper vector (Life Technologies) were transformed according to the manufacturer’s protocol with expression vectors containing the different gene constructs. Recombinant bacmids were selected through PCR screening, grown, and isolated. Subsequently, Sf9 cells (4 x 10⁵ cells*ml) in 2 ml of Insect-Xpress medium (Lonza, Walkersville, MD, USA) were transfected with recombinant bacmids using Cellfectin reagent (Life Technologies). After a three-day incubation period, recombinant baculoviruses were isolated (P1) and used to infect fresh Sf9 cells (1.2 x 10⁶ cells*ml) in 10 ml of Insect-Xpress medium (Lonza, Walkersville, MD, USA) with 15 mg/ml gentamycin (Roth, Karlsruhe, Germany) at a multiplicity of infection of 0.1. Five days after infection, the amplified viruses were harvested (P2 stock).

Mohammadi S., Herrera-Álvarez S., Yang L., Rodríguez-Ordoñez M.D., Zhang K., Storz J.F., Dobler S., Crawford A.J, & Andolfatto P. (2022). Constraints on the evolution of toxin-resistant Na,K-ATPases have limited dependence on sequence divergence. PLoS Genetics, 18(8), e1010323.

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Baculovirus Expression System Protocol

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Escherichia coli DH10bac cells harboring the baculovirus genome (bacmid) and a transposition helper vector (Life Technologies) were transformed according to the manufacturer’s protocol with expression vectors containing the different gene constructs. Recombinant bacmids were selected through PCR screening, grown, and isolated^{69 (link)}. Subsequently, Sf9 cells (4 x 10⁵ cells*ml) in 2 ml of Insect-Xpress medium (Lonza, Walkersville, MD, USA) were transfected with recombinant bacmids using Cellfectin reagent (Thermo Fisher). After a three-day incubation period, recombinant baculoviruses were isolated (P1) and used to infect fresh Sf9 cells (1.2 x 10⁶ cells*ml) in 10 ml of Insect-Xpress medium (Lonza, Walkersville, MD, USA) with 15 mg/ml gentamycin (Roth, Karlsruhe, Germany) at a multiplicity of infection of 0.1. Five days after infection, the amplified viruses were harvested (P2 stock).

Mohammadi S., Yang L., Harpak A., Herrera-Álvarez S., Rodríguez-Ordoñez M.D., Peng J., Zhang K., Storz J.F., Dobler S., Crawford A.J, & Andolfatto P. (2021). Concerted evolution reveals co-adapted amino acid substitutions in frogs that prey on toxic toads. Current biology : CB, 31(12), 2530-2538.e10.

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