All recombinant plasmids and yeast strains were generated using standard molecular biology techniques and are listed in the key resources table . Plasmids were constructed using the DH5α strain of E. coli (New England Biolabs). Arf1 constructs were purified from the Rosetta2 strain of E. coli (Novagen). E. coli strains were cultured in LB and TB media. Yeast cell viability assays and yeast cell imaging was performed as described below using S. cerevisiae strains listed in the key resources table . S. cerevisiae was cultured in standard yeast synthetic dropout media. P. pastoris strains used for expression of Gea2 constructs were cultured in BMGY media and are listed in the key resources table .
Dh5α strain of e coli
Manufactured by New England Biolabs
Sourced in United States
The DH5α strain of E. coli is a commonly used host strain for cloning and plasmid propagation. It is a non-pathogenic laboratory strain of the bacterium Escherichia coli.
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Lab products found in correlation
4 protocols using dh5α strain of e coli
1
Molecular Cloning and Yeast Cell Culture Protocols
2
Molecular Cloning and Protein Expression
3
Molecular Cloning and Yeast Cell Culture Protocols
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All recombinant plasmids and yeast strains were generated using standard molecular biology techniques and are listed in the key resources table . Plasmids were constructed using the DH5α strain of E. coli (New England Biolabs). Arf1 constructs were purified from the Rosetta2 strain of E. coli (Novagen). E. coli strains were cultured in LB and TB media. Yeast cell viability assays and yeast cell imaging was performed as described below using S. cerevisiae strains listed in the key resources table . S. cerevisiae was cultured in standard yeast synthetic dropout media. P. pastoris strains used for expression of Gea2 constructs were cultured in BMGY media and are listed in the key resources table .
4
Cloning and Characterization of NRRS Gene Promoters
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The promoter regions (1.5 kb upstream of the start codon) of NRRS genes, At1g74770 and At2g18140, were amplified from the Arabidopsis genomic DNA using the gene specific oligonucleotides (Supplementary Table 1 ), with flanking restriction sites, BamHI and SalI. The amplified products were eluted from gel using Pure Link Gel Extraction kit (Invitrogen, USA), quantified and digested with BamHI and SalI. The 1.5 kb BamHI/SalI promoter fragment was cloned upstream of the GUS gene, using T4 DNA ligase (NEB, USA), with linearized BamHI/SalI digested pORE-R2 (Coutu et al., 2007 (link)) vector and transformed to DH5-α strain of E. coli (NEB, Massachusetts, USA). The prm::GUS fusion constructs were validated by nucleotide sequencing and introduced into Agrobacterium tumefaciens (Smith and Townsend, 1907 (link)) strain GV3101. Arabidopsis plants were transformed using the floral dip method (Clough and Bent, 1998 (link)). The primary transformants were selected on medium containing kanamycin (50 μg/ml) and further grown to develop T3 seeds. For each promoter, five independent Arabidopsis transgenic lines were tested for their response to nematode infection.
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