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Southern blot hybridisation

JA José Mariano Amabis
EG Eduardo Gorab
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DNA extraction was carried out according to standard protocols43. DNA samples (5–7 µg of each sample per lane) were quantified by spectrophotometry (NanoDrop ND-100, Uniscience) three times prior to digestion with EcoRI. Restriction enzymes and DNA size markers were purchased from New England Biolabs. DNA from gels was alkaline transferred to Hybond N + membranes according to standard procedures (GE Healthcare). Three hybridisation assays were carried out overnight at 60 °C in 0.5 M Na2HPO4, 2% SDS. Ribosomal DNA41 and histone gene44 inserts were labelled by random priming with ⍺32P-dATP following current protocols (Life Tech). The membranes were washed twice at 60 °C for 30 min in 40 mM Na2HPO4, 2% SDS. Signal quantification was performed with ImageJ 1.48 V Analysis Software (Wayne Rasband, NIH, USA).

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