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View additional product information for MAGnify™ Chromatin Immunoprecipitation System - FAQs (492024)
12 product FAQs found
For each ChIP reaction, we recommend using 10,000-300,000 cells or 0.167-5 mg of tissue. To ensure consistency and decrease experimental variability, we recommend preparing a common chromatin batch suitable for multiple ChIP experiments. Note that following lysis, samples are at a concentration of 1 million cells/50 µL.
There might be excess chromatin or antibody added to the IP, or insufficient amount of DNA template added into the PCR reaction. Also your PCR conditions might need optimizing. Try decreasing the number of amplification cycles in PCR. Finally, it is ideal to have duplicate or triplicate runs for each IP to identify any issues, like human or product errors.
The most possible cause is the antibody does not function in IP. Not all antibodies used for western blotting will work well in ChIP. You need to verify the antibody is qualified for ChIP or IP applications. And try adding more antibody to the IP reaction and more DNA template to the PCR.
This is likely to be caused by insufficient chromatin amount in the IP reaction or insufficient antibody incubation time. We also recommend optimizing the crosslinking condition, and monitoring sonication of nuclei by microscope to ensure complete lysis.
First make sure that the PCR condition is fully optimized and check the primer design. Then try increasing the amount of template DNA added to the PCR reaction. Finally, evaluate sonication of nuclei by microscope to ensure complete lysis.
Shearing of the chromatin into appropriate fragments is required to ensure optimal ChIP results. Ideal shearing should yield DNA fragments between 200 and 1000 bp in length. ~200-500 bp fragments are usually used in downstream qPCR analysis and ~100-300 bp fragments are used in massive parallel DNA sequencing downstream analysis.
Yes. Please refer to this paper for chromatin preparation protocol from FFPE samples: Fanelli M et al. Nature Protocol, 2011; 6:1905-1919. Our R&D has collaborated with the authors to confirm that the MAGnify Chromatin Immunoprecipitation (ChIP) System is compatible with this protocol.
For fixed but not embedded samples (e.g., 10 min in 1% PFA), the standard protocol will work. No further modification is needed.
Yes. The MAGnify Chromatin Immunoprecipitation (ChIP) System has been validated for all major next-generation sequencing platforms. Usually 1-10 ng ChIP DNA is enough for next-generation sequencing. For next-generation sequencing support, please visit our Next-Generation Sequencing Support Center (thermofisher.com/ngssupport).
We recommend the following controls:
- Negative control antibody: Either do not use a primary antibody, or use the normal rabbit IgG or mouse IgG isotype control.
- Positive control antibody: This control ensures that each step of the procedure is working. For example, we observe consistent enrichment of heterochromatin markers such as H3-K9Me3 at the satellite repeat locus (SAT-2).
- Negative control PCR primer: This control is designed against a sequence that would not be enriched by your chromatin IP procedure.
- Input DNA control: Input DNA is DNA obtained from chromatin that has not been immunoprecipitated and has been reversed crosslinked similar to your samples. It is a control for PCR effectiveness and utilized in ChIP sequencing data analysis.
Both sonication and enzyme digestion are widely used for ChIP. Micrococcal nuclease results in greater reproducibility and allows better control of the fragment sizes. However, it can take more optimization and the enzyme can be compromised and fail to work properly.
Many factors influence yield, including cell type, target protein, antibody, etc. Generally speaking, anywhere between 0.5 ng to >10 ng can be obtained per ChIP reaction. Yields are usually too low to be measured using a NanoDrop spectrophotometer.
For each ChIP reaction, the MAGnify Chromatin Immunoprecipitation (ChIP) System recommends using 10,000-300,000 cells or 0.167 - 5 mg of tissue. The Thermo Scientific Agarose ChIP Kit and Thermo Scientific Magnetic ChIP Kit recommend 2 - 4 x 10E6 cells per ChIP reaction. To ensure consistency and decrease experimental variability, we recommend preparing a common chromatin batch suitable for multiple ChIP experiments.