Integrating vectors were digested with AseI and concentrated by ethanol precipitation. Transformations were conducted as previously described (2 (link)), with 3 μg of DNA and 30 μg of blocking DNA (Ultrapure salmon sperm DNA - Invitrogen). Transformants were plated using the top agar method on 100 μg/ml Hygromycin B and grown under 100 μmol m−2 s−1 for 3 weeks. Individual lines were transferred to 500 μl of F/2 with 100 μg/ml Hygromycin B (GoldBio) in a 96 deep well plate (Evergreen Biotech). Cultures were then maintained on solid plates.
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Microalgae Transformation Protocols
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Corresponding Organization : Michigan State University
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Variable analysis
independent variables
- Amount of DNA used for transformation (3 μg)
- Transformant growth and survival on Hygromycin B selection plates
- Blocking DNA concentration (30 μg of salmon sperm DNA)
- Hygromycin B concentration (100 μg/ml)
- Light intensity (100 μmol m^-2 s^-1)
- Growth duration (3 weeks)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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