How you lyse cell for Chelex bead DNA extraction
The correct answer and explanation is :
Cell lysis for Chelex bead DNA extraction is a critical step in obtaining pure DNA from biological samples. The Chelex method is a quick, inexpensive, and reliable technique for isolating DNA, especially for PCR amplification, from a variety of sources like blood, tissue, or cells.
Step-by-Step Cell Lysis for Chelex Bead DNA Extraction
- Cell Collection and Preparation:
- Collect the sample (e.g., blood, tissue, or cultured cells) and ensure it is in a suitable buffer, such as phosphate-buffered saline (PBS) if needed.
- For blood samples, a small volume (typically 200 µL) is taken.
- Incubation with Chelex Resin:
- Add a suspension of Chelex resin beads to the sample. Chelex 100, a chelating agent, is usually prepared at a concentration of 5-10%.
- The Chelex resin binds to metal ions such as magnesium and calcium, which are crucial for the activity of DNases. This prevents DNA degradation by inhibiting DNase activity.
- Heat Treatment for Cell Lysis:
- After adding the Chelex resin, the sample is heated at around 95°C for 10-15 minutes. This heat treatment disrupts the cell membrane and denatures proteins, including DNases, releasing the DNA into the solution.
- The heat also helps to break down the cellular components, like proteins and lipids, facilitating the extraction of DNA.
- Centrifugation:
- Following heat treatment, centrifuge the sample to pellet the Chelex beads and cellular debris.
- The supernatant, now containing the DNA, is carefully collected and used for downstream applications such as PCR.
Explanation of the Process
Chelex resin functions as a chelating agent that binds divalent metal ions (e.g., Mg²⁺, Ca²⁺), which are essential cofactors for DNase activity. By removing these metal ions, Chelex prevents the DNA from being degraded during the extraction process. The heat treatment is essential for lysing the cells and denaturing proteins, thereby releasing the DNA into solution. The centrifugation step separates the Chelex resin and debris from the DNA, leaving a purified DNA sample for analysis or amplification.
This method is widely used due to its simplicity and minimal requirements, making it suitable for applications like forensic analysis, genetic testing, or population studies.