No Product in Histone H3 Positive Control IP (ChIP (CST Guide))

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Positive control Histone H3-IP with RPL30 primer set produces no PCR product, indicating fundamental problems with IP procedure or elution. Common causes: 1) Insufficient chromatin added to IP reaction (<5 µg) 2) Insufficient antibody added to IP reaction (<10 µl Histone H3 antibody) 3) IP incubation time too short (less than overnight plus 2 hr with Protein G beads) 4) Incomplete elution of chromatin from Protein G beads due to suboptimal temperature or insufficient mixing Solutions: 1) Add 5-10 µg of chromatin per IP reaction 2) Add 10 µl of Histone H3 antibody to each IP reaction 3) Incubate with antibody overnight, then add Protein G beads and incubate additional 2 hr 4) Perform elution at 65°C with frequent mixing to keep beads suspended in solution