Rediprime 2 random labeling system

Ten micrograms of genomic DNA were digested with KpnI and separated by 0.8% agarose gel electrophoresis. The separated DNA was blotted onto a Hybond N⁺ nylon membrane (Amersham Biosciences, Piscataway, NJ, USA). A 0.3-kb PCR fragment corresponding to the C-terminal region of CrFAD7 was used as a probe. ³²P-labeled probes were produced using the Rediprime^TM II Random Labeling System (Amersham Biosciences), and hybridization was performed according to the manufacturer’s instructions. Signals were detected using the Bio-Imaging Analyzer BAS-1800II (Fuji, Tokyo, Japan).
Total RNA was extracted from C. reinhardtii cells (5 ml liquid culture) using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Twenty micrograms of total RNA were separated by gel electrophoresis and blotted onto a Hybond-N nylon membrane (Amersham Biosciences, USA) by capillary transfer. A 0.3-kb fragment corresponding to the CrFAD7 cDNA was used as a probe. Procedures for hybridization and signal detection were as described above.

For Southern blot analysis of the CpSRP43 and ChlM mutants, 10 μg of genomic DNA was digested with Pst I/NcoI and NcoI, respectively, separated by 0.8% agarose gel electrophoresis, and then blotted onto Hybond-N⁺ nylon membranes (Amersham Biosciences, Sweden). 0.3-kb PCR fragments corresponding to the aphVII, CpSRP43 and ChlM genes were used as probes. The primers used to generate each probe are listed in Table S1. ³²P-labeled probes were produced using the Rediprime^TM II Random Labeling System (Amersham Biosciences, Sweden) and hybridization was performed following the manufacturer’s instructions. The probe-bound membrane was washed and exposed to a phosphor-imaging plate (IP) for 3 days, and the obtained signals were detected using a Bio-Imaging Analyzer BAS-1800II (Fuji, Japan).