Hybond n nylon membrane

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.

RNA was extracted with a NucleoSpin RNA kit (Macherey-Nagel) according to the manufacturer’s instructions. Northern blotting was performed as previously described (39 (link)). In brief, 0.2 μg of RNA was denatured and separated by an 0.8% agarose-formaldehyde gel at 70 V for 5 h. The agarose gel was soaked in 50 mM NaOH for 50 min to break the large RNA fragment. Subsequently, the gel was washed with 100 mM Tris-HCl (pH 7.5) for 30 min and incubated in 20× SSC buffer (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) for 20 min and then capillary transferred to a positively charged Hybond-N nylon membrane (Amersham Biosciences) overnight. RNA was immobilized by UV cross-linking (1,800 × 100 μJ/cm) and hybridized at 50°C overnight with digoxigenin (DIG)-labeled probes generated with a PCR DIG probe synthesis kit (Roche Diagnostics). The primers used to synthesize the DIG-labeled nCoV19-N cDNA probe were 5′-AAGCTGGACTTCCCTATGGTGC-3′ and 5′-CCTTGGGTTTGTTCTGGACCACG-3′. The probes of porphobilinogen deaminase (PBGD) used as the internal control in Northern blotting were 5′-GGTGACCAGCACACTTTGGG-3′ and 5′-AGCCGGGTGTTGAGGTTTCC-3′.