What is the ChIP method?

Chromatin immunoprecipitation (ChIP) is a type of immunoprecipitation experimental technique used to investigate the interaction between proteins and DNA in the cell. The DNA-protein complexes (chromatin-protein) are then sheared into ~500 bp DNA fragments by sonication or nuclease digestion.

What are the four steps of DNA purification?

DNA Purification Basics

  • Creation of Lysate. The first step in any nucleic acid purification reaction is releasing the DNA/RNA into solution.
  • Clearing of Lysate.
  • Binding to the Purification Matrix.
  • Washing.
  • Elution.

How is DNA purification done?

Basically, you can purify your DNA samples by lysating your cell and/or tissue samples using the most appropriate procedure (mechanical disruption, chemical treatment or enzymatic digestion), isolating the nucleic acids from its contaminants and precipitating it in a suitable buffer solution.

What does proteinase K do in ChIP?

Samples are treated with proteinase K, which cleaves peptide bonds adjacent to the carboxylic group of aliphatic and aromatic amino acids. Cross-links between proteins and DNA are disrupted which aids DNA purification. Purify DNA using a PCR purification kit or phenol:chloroform extraction.

What is ChIP analysis used for?

The ChIP assay has become a very popular tool for studying chromatin structure and nuclear events involved in transcription. It has been used to identify target genes of many important DNA-binding proteins and their regulatory enzymes.

Why is ChIP useful?

Why is ChIP such a useful technique? ChIP dissects the spatial and temporal dynamics of the interactions between chromatin and its associated factors. The technique allows us to map minute-by-minute changes at a single promoter or follow a single transcription factor over the entire human genome.

What is the difference between DNA extraction and DNA purification?

Extraction makes use of a solvent that serves as the extractant and has two stages: (i) gentle lysis of the cells / solubilization of DNA and (ii) removal of contaminants (proteins, RNA and other macromolecules) or the so-called purification is achieved either by enzymatic or chemical means.

Why is DNA purification needed?

Why DNA purification is important DNA purification helps extract genomic and/or plasmid DNA in the sample quantities that your research requires. Purifying your DNA samples from contaminants also extends their shelf-life and reduces the probability of error when it comes to research results.

Why do we add proteinase K to our DNA extractions?

Why is this enzyme used in DNA extraction? Proteinase K is used during DNA extraction to digest many contaminating proteins present. It also degrades nucleases that may be present in DNA extraction and protects the nucleic acids from nuclease attack.

What do you need to know about Chip immunoprecipitation?

After sonication, pellet cell debris by centrifugation for 10 min, 4°C, 8,000 x g. Transfer supernatant to a new tube. This chromatin preparation will be used for the immunoprecipitation (IP) in Step 4. Remove 50 μL of each sonicated sample, to determine DNA concentration and fragment size.

How is sonicated chromatin used for Chip immunoprecipitation?

Transfer supernatant to a new tube. This chromatin preparation will be used for the immunoprecipitation (IP) in Step 4. Remove 50 μL of each sonicated sample, to determine DNA concentration and fragment size. The sonicated chromatin can be snap frozen in liquid nitrogen and stored at -80°C for up to 3 months. Avoid multiple freeze-thaws.

How are suspension cells used in chip treatment?

When using suspension cells, start with 1×107–​5×107 cells and treat with both 0.75% formaldehyde and glycine as described above (Step 1). Pellet cells by centrifugation (5 mins, 1,000 x g).

How are probes and primers designed for Chip?

Primers and probes are often designed using software provided with the real-time PCR apparatus or using an online design tool. A selection of pre-designed primers and probes is available on our website. Alternatively, analysis can be by sequencing when using the ChIP-seq method.