Immunoprecipitation and Quantitative PCR

Meiotic cells were processed as described [65 (link)], with the following modifications: Lysis was performed in Lysis buffer plus 1 mM PMSF, 50 μg/mL Aprotinin and 1X Complete Mini EDTA-Free (Roche), using 0.5 mm zirconium/silica beads (Biospec Products, Bartlesville, OK). 2 μg of the mouse monoclonal anti-FLAG antibody M2 (Sigma) and 30 μL Protein G magnetic beads (New England Biolabs) were used. Quantitative PCR was performed from the immunoprecipitated DNA or the whole-cell extract using a 7900HT Fast Real-Time PCR System (Applied Biosystems, Thermo Scientific) and SYBR Green PCR master mix (Applied Biosystems) as described [65 (link)]. Results were expressed as % of DNA in the total input present in the immunoprecipitated sample. Primers for GAT1, BUD23, ERG1, Axis and NFT1 loci have been described [50 (link), 51 (link), 66 (link)].