ChIP Protocol

critical

No Product in Histone H3 Positive Control IP

Positive control Histone H3-IP with RPL30 primer set produces no PCR product, indicating fundamental problems with IP procedure or elution.

💡 4 · ✓ 4

critical

No or Minimal PCR Product in Input Control

Input chromatin PCR reactions produce no product or very little product, indicating problems with DNA quantity, PCR conditions, or primer design.

💡 5 · ✓ 5

severe

DNA Fragment Size Too Large for Resolution

ChIP-seq peaks are broad and poorly defined, with low spatial resolution for pinpointing binding sites. Background signal is elevated across large genomic regions.

💡 4 · ✓ 5

severe

Low ChIP Signal Due to Chromatin Over-Fragmentation

Low or absent signal in ChIP experiment despite proper antibody and starting material. Chromatin fragments are smaller than 500 bp after sonication or enzymatic digestion.

💡 4 · ✓ 4

severe

Low Enrichment Due to Insufficient Starting Material

ChIP-seq exhibits low resolution with high background across large genomic regions. Signal-to-noise ratio is poor, with diffuse peaks and elevated baseline signal throughout the genome.

💡 4 · ✓ 4

severe

Chromatin Under-Fragmentation with Excessive Large Fragments

Chromatin fragments are too large (>900 bp for enzymatic, >1 kb for sonication), leading to increased background signal and lower resolution in ChIP results.

💡 4 · ✓ 4

severe

Chromatin Over-Fragmentation to Mono-Nucleosome Length

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.

💡 4 · ✓ 4

severe

Fragmented Chromatin Concentration Below Required Threshold

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.

💡 4 · ✓ 4

moderate

Loss of Specific Signal Due to Overly Stringent Wash Conditions

Low signal at expected target regions while background is also very low. Positive control regions show reduced signal compared to expected levels.

💡 4 · ✓ 4

moderate

Low ChIP Signal from Ineffective Cell Lysis

Overall low signal with visible cell clumps or debris in lysate. Chromatin yield is lower than expected based on starting cell number.

💡 4 · ✓ 4

moderate

Low ChIP Signal from Insufficient Starting Material or Antibody

Weak or barely detectable signal across all samples including positive controls. Signal intensity is uniformly low rather than selectively absent at specific regions.

💡 4 · ✓ 4