Decaprime 2 kit

Total RNA from the cells of colonies grown on GMA plates was isolated by the hot phenol method as previously described [14 (link)]. For northern blots, 12 μg of total RNA was separated on a 1.5% agarose gel, transferred to a positively charged nitrocellulose membrane (Hybond-XL, Amersham Bioscience) and exposed to a labeled probe. The radioactive signal was visualized on Fuji X-ray film. The DNA probe for the RDN18 gene was a complete ORF of the gene prepared by PCR reaction. For the FLO11 probe, a PCR fragment corresponding to the last 1382 bp of the FLO11 gene was used. The [α-³²P] dCTP-labeled probes were obtained by random priming using the DecaPrime II Kit (Ambion).

We generated a probe for EGFP mRNA by PCR-amplifying the complete EGFP gene in pEGFP-N1. The probe was radiolabeled using the DECAprime II Kit (Ambion, Austin, TX) and ³²P-dCTP, according to the manufacturer's instructions. RNA was extracted from cultured cells with the RNeasy Mini Kit (QIAGEN) and subjected to electrophoresis on a 1% agarose denaturing formaldehyde gel (2.2 M formaldehyde; 200 mM MOPS, pH 7.0; 50 mM sodium acetate; 10 mM EDTA). The products were then transferred to a Amersham Hybond N+ nylon membrane (GE Healthcare, Piscataway, NJ) overnight and hybridized with the labeled probe, using ULTRAhyb Ultrasensitive Hybridization Buffer (Ambion).

Northern blotting was performed essentially as described [132 (link)]. In brief, 20 μg of yeast total RNA was prepared in Glyoxal sample load dye (Ambion) and separated by 1% agarose gel electrophoresis. RNA was transferred on to membrane by capillary blotting for pre-hybridization. Pre-hybridization solution contained 50% formamide, 10% Dextran sulfate, 5× Denhardt’s solution, 1 M NaCl, 50 mM Tris-HCl pH 7.5, 0.1% SDS, 0.1% sodium pyrophosphate, and 500 μg/ml denatured salmon sperm DNA. DNA double-stranded probes were generated by PCR and radiolabeled with ³²P-dATP using the Decaprime II kit (Ambion) according to the manufacturer’s instructions. Blots were hybridized over night at 42 °C and washed twice each in 2× SSC for 15 min at 42 °C, in 5× SSC with 0.5% SDS for 30 min at 65 °C, and in 0.2× SSC for 30 min at room temperature. Blots were visualized by phosphorimaging (Bio-Rad or GE Healthcare) and quantified using Quantity One (Bio-Rad).

Mouse retina cDNA was prepared as described [135] (link). vps26A was PCR-amplified using primers 5′-GAGTTTTCTTGGAGGCTTTTTTGGTCC-3′ and 5′-TTACATCTCAGGCTGCTCCGCAGAGG-3′ and a 30 ng cDNA template. The PCR product was cloned into pBluescript by blunt-end ligation and verified by sequencing. DNA probes were generated from gel-purified insert (excised with EcoRI and HindIII) by random-prime labeling with [α-³²P]dCTP using the DECAprimeII kit (Ambion). Unincorporated nucleotides were removed with a MicroBio-Spin 30 column (Bio-Rad).
For Northern blot analysis, total RNA was prepared from retinas of 3–4-mo-old CD-1 mice by homogenization in TRI reagent (Ambion) using a motorized pestle and passage through a 20-gauge needle, extraction with 1-bromo-3-chloro-propane, and purification with the RNeasy kit (Qiagen). We resolved 10 µg of total RNA on a formaldehyde-agarose gel and transferred it to a Hybond-N+ nylon membrane (GE Healthcare) by capillary transfer. Membranes were prehybridized in ULTRAhyb (Ambion) for 1 h, then incubated at 42°C overnight with a ∼2.5×10⁷ cpm denatured probe diluted in ULTRAhyb. Blots were washed several times in 2× SSC+0.1% SDS and several times in 0.1× SSC+0.1% SDS. Washes were performed at 42–45°C. For imaging, blots were exposed to a storage phosphor screen and scanned on a Typhoon (GE Healthcare).