Chapter 12: DNA Extraction From Cell culture

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DNA extraction from cultured cells is a routine procedure in many research laboratories, enabling scientists to investigate cellular processes, gene expression, and genetic variations. The spin column method offers a fast and efficient way to isolate high-quality DNA from cultured cells. This blog post will guide you through the typical steps involved in this process.

Materials Required

  • Cultured cells
  • Lysis buffer
  • Proteinase K
  • RNase A
  • Binding buffer
  • Wash buffer
  • Elution buffer
  • Spin columns
  • Collection tubes
  • Microcentrifuge

Procedure

  • Cell Harvesting and Lysis:
    • Harvest the cultured cells from the culture dish or flask.
    • Add lysis buffer to the cell pellet. This buffer contains detergents and enzymes, like Proteinase K, that break down cell membranes and proteins, releasing DNA into the solution.
    • Incubate the sample at an appropriate temperature to ensure complete lysis.
  • RNA Removal:
    • Add RNase A to the lysate to degrade RNA, which can interfere with downstream applications.
  • DNA Binding to Spin Column:
    • Add binding buffer to the lysate. This buffer creates conditions that promote DNA binding to the silica membrane within the spin column.
    • Transfer the lysate to a spin column.
    • Centrifuge the column to bind DNA to the membrane and remove impurities.
  • Washing:
    • Wash the spin column with wash buffer to remove any remaining contaminants.
    • Centrifuge the column to remove the wash buffer.
  • DNA Elution:
    • Add elution buffer (low salt buffer or water) to the spin column.
    • Centrifuge the column to elute the purified DNA into a collection tube.
  • Tips and Considerations
  • Use appropriate cell harvesting techniques for your specific cell type.
  • Ensure all reagents and equipment are RNase-free to prevent RNA contamination.
  • Optimize lysis and binding conditions for efficient DNA extraction.
  • Handle cell cultures and reagents with care and follow appropriate safety guidelines.

Applications of Extracted DNA from Cultured Cells

The DNA extracted using the spin column method from cultured cells can be used for various applications, including:

  • Gene expression analysis
  • Genotyping
  • DNA sequencing
  • Mutation analysis
  • Transfection and gene editing experiments

Conclusion

Spin column-based DNA extraction from cultured cells is a convenient and reliable technique widely used in research laboratories. By following the outlined steps and considering the provided tips, you can successfully isolate high-quality DNA from cultured cells for your downstream applications.

Lysis Buffer : DNA lysis buffer is a solution that breaks open  cells to extract DNA. It’s used in molecular biology experiments to analyze DNA.

Binding Buffer : A binding buffer is a reagent used to bind DNA to silica during DNA extraction. It helps to purify DNA from various samples, including blood, cells, and tissues. It contains Guanidine Hydrochloride and Nuclease free water

Washing buffer :DNA Wash Buffer (80% ethanol, 20 mM NaCl, 2 mM Tris-HCl, pH 7.5)

Elution buffer: (0.5 mM EDTA, 10 mM Tris-HCl, pH 9.0 )

 DNA Extraction Protocol

  • Centrifuge the appropriate number of cells (maximum 5 x 106) at 13,000 rpm for 10 sec( or 800 rpm for 5 min). Resuspend the pellet in 200 μl PBS. Add 200μl of Lysis buffer + 20μl Proteinase K +10 μl Rnase Aand Mix thoroughly by vortexing.
  • Incubate at 65°C for 15-20 minutes either on thermo mixer or water bath or dry bath. Briefly invert the tube once during incubation.
  • After incubation add 200μl Isopropanol to the lysate. Mixed by inverting a tube 15- 20 times.
  • Transfer entire lysate to the DNA spin column, and centrifuge at 12,000 rpm for 2 mins. Discard the flow through liquid 
  • Repeat above step, until the entire sample has been processed and retain column for further processing. 
  • Place the column into the same collection tube. Add 500μl of Binding buffer. Centrifuge at 12,000 rpm for 2 min. discard the flow through.
  • Place the column into same collection tube. Add 500μl of Wash buffer. Centrifuge at 12,000 rpm for 1 min. discard the flow through.
  • Place empty DNA spin column, with the lid open into the same collection tube and centrifuge at 12,000 rpm for 2 min.
  • Place the column into a new sterile 1.5 ml eppendorf tube, add 30 μl preheated Elution buffer. Incubate at room temperature for 5 min. (perform this step twice 30 μl + 30 μl = 60 μl)
  • Centrifuge 10,000 rpm for 1 min to elute pure blood gDNA. The first elution normally yields 60-70 % of DNA bound. A second elution with another 30 μl buffer will yield another 20 % of the DNA.
  • Note: Elution volume may vary as per downstream process
  • Discard the Column, and save elute. Do not reuse binding columns or collection tubes.

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