pMHL_2002-bfloGFPa1

Protocol tips

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	The protocol for diatom transformation was adapted from Poulsen et al. (2006). Axenic exponential-phase wild-type T. pseudonana cells (1 × 108 cells total) were pelleted (3000×g for 10 min) and plated on ASW medium 1.2% agar plates. M-17 tungsten particles were coated with 5 μg of plasmid DNA for each construct with the CaCl2-spermidine method as per the manufacturer’s instructions (Bio-Rad, 165-2267).

Protocol tips

The protocol for diatom transformation was adapted from Poulsen et al. (2006). Axenic exponential-phase wild-type T. pseudonana cells (1 × 108 cells total) were pelleted (3000×g for 10 min) and plated on ASW medium 1.2% agar plates. M-17 tungsten particles were coated with 5 μg of plasmid DNA for each construct with the CaCl2-spermidine method as per the manufacturer’s instructions (Bio-Rad, 165-2267).

Publication protocol

The protocol for diatom transformation was adapted from Poulsen et al. (2006). Axenic exponential-phase wild-type T. pseudonana cells (1 × 108 cells total) were pelleted (3000×g for 10 min) and plated on ASW medium 1.2% agar plates. M-17 tungsten particles were coated with 5 μg of plasmid DNA for each construct with the CaCl2-spermidine method as per the manufacturer’s instructions (Bio-Rad, 165-2267). Each plate was bombarded twice with 2–5 mg of coated tungsten beads using the Biolistic DS-1000/He particle delivery system with a 1350 psi rupture disk. Following bombardment, cells were immediately resuspended in 10 ml of ASW medium and incubated for 24 h under constant illumination (150 μE m−2 s−1). The following day, the cell density was determined and a range of concentrations (1 × 106–5 × 106 cells) were plated on ASW agar plates containing the antibiotic nourseothricin at a final concentration of 100 μg ml−1. The plates were incubated at 18 °C in continuous light for approximately 2 weeks. Individual colonies from the plates were isolated and screened by PCR and fluorescence microscopy to confirm nuclear insertion of the plasmid and for GFP expression, respectively.

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Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Protein Expression Eukaryotic cells - T. pseudonana IbpA DR2 antigen from Histophilus somni using pMHL_2002-bfloGFPa1 from Mark Hildebrand, Marine Biology Research Division, Scripps Insti.

Manufacturer protocol

Download the product protocol from Mark Hildebrand, Marine Biology Research Division, Scripps Insti for pMHL_2002-bfloGFPa1 below.

Videos

Check out videos that might be relevant for performing Protein Expression Eukaryotic cells - T. pseudonana IbpA DR2 antigen from Histophilus somni using pMHL_2002-bfloGFPa1 from Mark Hildebrand, Marine Biology Research Division, Scripps Insti. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.