Cloning Brassica napus Gene into Arabidopsis

Genomic DNA from Brassica napus was amplified using Phusion Proofreading polymerase (Thermo Fischer) and primers with specific restriction sites. The amplified DNA was separated on an agarose gel and extracted using a GeneJet gel extraction kit (Thermo Fischer) and then ligated into a pJET2.1 cloning vector using the CloneJet kit (Thermo Fischer). The insert was digested and separated on an agarose gel and then cloned into a pHPT1 binary vector [44 (link)], using T4 Ligase (Thermo Fischer). The resulting construct was transformed into Agrobacterium and then into Arabidopsis srr1–1 plants using the floral dip method.

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Schiessl S., Williams N., Specht P., Staiger D, & Johansson M. (2019). Different copies of SENSITIVITY TO RED LIGHT REDUCED 1 show strong subfunctionalization in Brassica napus. BMC Plant Biology, 19, 372.

Publication 2019

Phusion Proofreading polymerase GeneJet gel extraction kit CloneJet kit T4 Ligase

Agarose Agrobacterium Arabidopsis Brassica napus Genomic Ligase Plants Primers Vector

Corresponding Organization :

Other organizations : University of Giessen, Bielefeld University

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Variable analysis

independent variables

Use of Phusion Proofreading polymerase (Thermo Fischer) for DNA amplification
Use of primers with specific restriction sites
Ligation of amplified DNA into pJET2.1 cloning vector using CloneJet kit (Thermo Fischer)
Digestion of insert and cloning into pHPT1 binary vector using T4 Ligase (Thermo Fischer)
Transformation of the resulting construct into Agrobacterium and then into Arabidopsis srr1–1 plants using the floral dip method

dependent variables

Growth and phenotypic characteristics of the transformed Arabidopsis srr1–1 plants

control variables

Use of Brassica napus as the source of genomic DNA
Use of Arabidopsis srr1–1 plants as the target for transformation

controls

No positive control is explicitly mentioned
No negative control is explicitly mentioned

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