Megaprime dna labelling system

Total RNA from CRC cells was purified. A ZFAS1-specific, radioactive DNA probe with a length of 300 bp was generated using [α-³²P]dCTP (Perkin Elmer) and the Megaprime DNA labelling system (GE Healthcare, Pittsburgh, PA, USA). Hybridization was performed using QuickHyb (Agilent, Santa Clara, CA, USA), following the manufacturer's instructions.

Fungal genomic DNA was isolated from mycelium as previously described [57 (link)] or using an Extract-N-Amp plant PCR kit (Sigma). Genomic digests were transferred to positively charged nylon membranes (Hybond-N⁺, GE healthcare) by capillary transfer and fixed by UV cross-linking in a UV cross-linker (CL-1000, Ultra-Violet Products) at 7 x 10⁴ μJ/cm². Filters were probed with knockout vector pNPP50 labeled with [α-³²P] dCTP (3000 Ci/mmol; Perkin Elmer) using a random-primed DNA labeling kit (Megaprime DNA Labelling System, GE Healthcare). Hybridizations were performed at 42°C for 20 h in 5 x SSPE (20x SSPE; 3 M NaCl, 173 mM NaH₂PO₄-2H₂O, 25 mM EDTA), 50% formamide, 5x Denhardt’s solution, 1% SDS and 100 μg/ml denatured salmon sperm DNA. Membranes were washed with 1 x SSPE and 0.2% SDS at 65°C for 10 min and then with 0.1 x SSPE and 0.1% SDS at 65°C for 5 min. The membrane was then subjected to autoradiography.

The plants were grown for 2 weeks on ½MS media with 1% sucrose, then transferred to soil and grown for 2 weeks at 157 µE m⁻² s⁻¹, 16 h light and 20°C/8 h dark and 19°C. Leaves were harvested 2 h after dawn. Polysome analysis was done as described previously (Barkan, 1993 (link)). The psaA (AtCg00350), psbA (AtCg00020), lhcA4 (AT3G47470) and lhcB1.2 (AT1G29910) probes were amplified from A. thaliana DNA using gene‐specific primers (see Supporting Information: Table S7 radioactively labelled with α³²P[CTP] using the Megaprime DNA Labelling System (GE Healthcare Life Sciences), and hybridised at 65°C.