∆abf2 and ∆abf2 mhr1-1 cells were cultivated in YPGly media, then transferred to RGal complete media and cultivated at 30 °C for two days. Total cellular DNA was then prepared and digested by ApaI. Approximately 15 µg of total DNA was separated by electrophoresis on a 1.0% agarose gel, run at 24 °C for 80 h at 5 V/cm, and transferred to a nylon membrane (Amersham Hybond N Plus; GE Healthcare). Signals for mtDNA were detected using 32P-labeled full-length mtDNA from budding yeast as a probe. Signals were analyzed using a Typhoon FLA 7000 biomolecular imager (GE Healthcare).
Amersham hybond n plus
Manufactured by GE Healthcare
Amersham Hybond N Plus is a laboratory equipment product designed for membrane-based nucleic acid hybridization and detection. It is a positively charged nylon membrane that provides efficient and reliable transfer and immobilization of nucleic acids.
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Lab products found in correlation
2 protocols using amersham hybond n plus
1
Mitochondrial DNA Extraction and Analysis
2
Quantifying mtDNA and Nuclear DNA
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Total cellular DNA (∼15 μg) was separated by electrophoresis on a 0.5% agarose gel, run at 4°C for 30 h at 1 V/cm, and transferred to a nylon membrane (Amersham Hybond N Plus; GE Healthcare). Signals for mtDNA were detected using the 32P-labeled mtDNA OH region as a probe, as described (Anderson et al., 1981 (link); Lehtinen et al., 2000 (link)). Signals for nuclear DNA were detected using a 32P-labeled DNA fragment of the β-actin gene, ACTB (for nuclear DNA), as a probe. Primers designed for the mtDNA OH region were forward, 5′-TAACCACTCACGGGAGCTCT-3′, and reverse, 5′-AAGGCTAGGACCAAACCTAT-3′. Primers designed for ACTB were forward, 5′-TGCGTGACATTAAGGAGAAGCTGTGC-3′, and reverse, 5′-CTCGTCATACTCCTGCTTGCTGATCC-3′. Signals corresponding to mtDNA and nuclear DNA fragments were quantitated using a Fujifilm BAS-2500 image analyzer (Fujifilm, Tokyo, Japan).
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