Nytran membrane

Northern blot analysis was done as described (Rak et al. 2007b (link)). The RNAs, extracted from whole cells grown in YPGalA, were separated in a 1% (w/v) agarose-6% (v/v) formaldehyde gel in MOPS buffer (20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA), and then transferred to a Nytran membrane (Schleicher & Schuell) and hybridized with DNA probes specific to ATP9 (a PCR product amplified from MR6 DNA with primers 5′-AATAAGATATATAAATAAGTCC and 5′-GAATGTTATTAATTTAATCAAATGAG) and 21S RNA (a PCR product amplified with primers 21S.1: 5′-GTATAAGGTGTGAACTCTGCTCCAT and 21S.2: 5′-GGGGAGACAGTTGTTGTATCATTAC). The ATP9 and 21S RNA probes were labeled with [α-32P]-dCTP using the Prime-a-Gene Labelling System kit from Promega and purified on MicroSpin G-25 columns from Amersham Biosciences. Hybridizations were carried out in 50% (v/v) formamide, 5× SSPE, 0.5% SDS, 7% polyethylene glycol 5000, 5× Denhardt’s solution, 100 µg/mL carrier DNA at 42°C.

Genomic DNA was isolated from mouse tail as previously described [7] (link). The DNA was digested overnight at 37°C with BamH I or Pst I, and separated on 0.7% or 0.9% agarose. The gel was denatured and neutralized prior to transferring overnight to a Nytran membrane (Schleicher & Schuell, Keene, NH). Using a vacuum oven pre-warmed to 80°C, the blot was baked for 2 h. Prior to hybridization with probe, the blot was prehybridized for 5 h at 42°C in hybridization buffer (5X SSPE, 50% formamide, 5X Denhardt's solution, 0.1% SDS, and 0.1 mg/ml denatured salmon sperm DNA). Using ³²P labeled iNOS or hMnSOD cDNA, the membrane was hybridized at 42°C for 48 h. Using a buffer containing 5X SSC, 0.1% SDS, and 0.05% sodium pyrophosphate, the blot was washed for 15 min at room temperature twice. The last washing step was performed at 65°C in 0.1X SSC, 0.1% SDS, and 0.05% sodium pyrophosphate. The blot was then exposed to Kodak x-ray film at −80°C overnight.