Problems with PCR amplification in ChIP

Possible causes

• Primers binding nonspecifically due to low annealing temperature
• Excess primer or excess ChIP template
• Primer-dimer formation

• Increase annealing temperature by 2–4°C.
• Reduce primer concentration (eg, from 500 nM → 200–300 nM).
• Reduce template DNA input; ChIP DNA is often highly enriched and can saturate reactions.
• Run products on a gel to verify size; redesign primers if needed.

• High background due to insufficient washing
• Antibody crossreactivity or nonspecific bead interactions
• Chromatin overfragmentation

• Increase wash stringency: include additional high-salt or LiCl washes.
• Confirm antibody specificity with an independent method (IP, western).
• Check fragmentation on a bioanalyzer or gel; avoid <100 bp smearing.

Possible causes

• Real-time PCR solutions may be contaminated, we suggest preparing new solutions from stocks.

• Be sure to include standard/input DNA to confirm that the primers are working well.