No or Minimal PCR Product in Input Control (ChIP (CST Guide))

Question

Accepted Answer

Input chromatin PCR reactions produce no product or very little product, indicating problems with DNA quantity, PCR conditions, or primer design. Common causes: 1) Insufficient DNA added to PCR reaction 2) PCR conditions not optimized for experimental primer set 3) PCR amplified region spans nucleosome-free region with insufficient DNA 4) Insufficient chromatin added to IP or chromatin over-fragmented to mono-nucleosome length 5) Amplicon length exceeds optimal range for fragmented chromatin Solutions: 1) Add more DNA to PCR reaction or increase number of amplification cycles 2) Optimize PCR conditions using purified DNA from cross-linked and fragmented chromatin 3) Design different primer set with amplicon length <150 bp to match chromatin fragment size 4) Add 5-10 µg chromatin per IP and verify fragment size is 150-900 bp (not predominantly mono-nucleosome) 5) Ensure amplicon does not span nucleosome-free regulatory regions