All strains used for the resection assay harbor a single I-PpoI cleavage site near the 5′ end of lys1. Meiosis was induced and DNA content was analyzed by flow cytometry as described (33 (link)) (Supplementary Figure S2). Three h after induction of meiosis, 3 μM anhydrotetracycline (ahTet) (Acros 13803–65–1) was added to the culture, and samples were harvested hourly for 4 h after addition of ahTet. Cells were embedded in agarose plugs, and DNA was extracted as described (28 ). DNA in agarose plugs was digested with XbaI overnight at 37°C, electrophoresed and transferred to a nylon membrane (Zeta-probe GT membrane, Biorad). A 1-kb PCR product (primers OL3240 and OL3241) labeled with [α-32P] dCTP (3000 Ci/mmol) was used as a probe to detect resected DNA, which hybridized 3–4 kb away from the I-PpoI cutting site (Figure 4A ). Signals were detected using a Typhoon storage PhosphorImaging system (GE Healthcare).
Typhoon storage phosphorimaging system
Manufactured by GE Healthcare
The Typhoon storage PhosphorImaging system is a laboratory equipment designed for the detection and quantification of radioactive signals in biological samples. The system utilizes a phosphor screen to capture and store the radioactive signals, which can then be scanned and digitized for further analysis.
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2 protocols using typhoon storage phosphorimaging system
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Meiotic DNA resection analysis
2
DNA Damage Analysis at ade6-3049 Hotspot
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Meiotic induction and DNA analysis were described by45 (link), 46 . DNA in agarose plugs was digested with BsrG1 overnight at 37 °C, subjected to electrophoresis through 0.8% agarose, and transferred to a nylon membrane (Zeta-probe GT membrane, Biorad). DNA broken at the ade6-3049 DSB hotspot was detected by hybridization to a 1 kb probe from the right end of the 10.1 kb BsrGI fragment. The probe was a PCR product amplified from wild-type genomic DNA using OL3693 and OL3694 as primers and labeled with [α-32P] dCTP (3000 Ci/mmol). Signals were detected using a Typhoon storage PhosphorImaging system (GE Healthcare).
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