Content.High background of non-specific control antibodies.Weak or no signal.Low resolution with large areas of high background.Problems of PCR amplification.Low DNA recovery.cell lysis.chromatin scissors.
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Chromatin immunoprecipitation protocol
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The Observation Troubleshooting Guide is intended to provide possible causes and solutions to identifiable problems observed when using Chromatin Immunoprecipitation (ChIP).
Too much/little communication stitching
Insufficient can potentially helpTo prevent the dissociation of protein-DNA complexes on the next path and lead to an unsuccessful exit.
Excessive cross-linking can mask epitope insertions important for antibody binding, prevent complete cleavage, and chromatin prevent successful cleavage of the complex in subsequent steps.
If you are using paraformaldehyde, make sure it is freshly prepared.
Do not use 1.7 ml microcentrifuge tubes for sample volumes greater than 400 µl.
Hold the ultrasonicator very close to the bottom of the distiller or probe.
Lack of chromatin
Cut out significantly more replicas, increase ultrasound power, reduce the number of meshes, or use significantly fewer cells.
Perform lower shear replications, reduce human ultrasound power, generate more or fewer cells.
Indeed, the samples must be somewhere between sonication steps. If the sonication is too long or too strong, unwanted denaturation may occur too.