Equivalent Signal in Negative IgG and Positive H3 Controls (ChIP (CST Guide))

Question

Accepted Answer

Quantity of PCR product in negative control Rabbit IgG-IP equals that in positive control Histone H3-IP, indicating high non-specific binding or PCR over-amplification. Common causes: 1) Excessive chromatin added to IP reaction (>15 µg) 2) Too much Histone H3 antibody added (>10 µl per IP) 3) Excessive normal rabbit IgG added to negative control (>1 µl per IP) 4) Too much DNA added to PCR reaction causing saturation 5) Excessive PCR cycles causing amplification beyond linear phase Solutions: 1) Add no more than 15 µg chromatin and 10 µl Histone H3 antibody per IP 2) Reduce normal rabbit IgG to 1 µl per IP reaction 3) Add less DNA to PCR reaction to stay within linear amplification range 4) Decrease number of PCR cycles to ensure quantitation within linear phase 5) Optimize PCR using serial dilutions of input DNA to determine linear range