Real lab failures, root causes, and fixes — curated and bilingually annotated by our team.
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.
Positive control Histone H3-IP with RPL30 primer set produces no PCR product, indicating fundamental problems with IP procedure or elution.
Input chromatin PCR reactions produce no product or very little product, indicating problems with DNA quantity, PCR conditions, or primer design.
Chromatin fragments are too large (>900 bp for enzymatic, >1 kb for sonication), leading to increased background signal and lower resolution in ChIP results.
More than 80% of DNA fragments are shorter than 500 bp, resulting in diminished PCR signal especially for amplicons >150 bp, and potential disruption of chromatin integrity and antibody epitopes.
Experimental antibody-IP PCR reaction produces no product while positive control H3-IP works, indicating antibody-specific or target-specific issues.
DNA concentration of chromatin preparation is insufficient for ChIP, falling below the recommended 50 µg/ml or unable to provide 5-10 µg per IP reaction.
DNA concentration of fragmented chromatin preparation is below expected ranges (e.g., <100 µg/ml for HeLa cells, <20 µg/ml for brain tissue). Insufficient material for recommended 5-10 µg chromatin per IP reaction.
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