Chapter 6: DNA Extraction From  Animal Tissue

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DNA extraction is a fundamental technique in molecular biology, enabling researchers to isolate and study genetic material. When working with animal tissue, a spin column method offers a convenient and efficient way to obtain high-quality DNA. This blog post will outline the general steps involved in DNA extraction from animal tissue using a spin column.

Materials Required

  • Animal tissue sample
  • Lysis buffer
  • Proteinase K
  • RNase A
  • Binding buffer
  • Wash buffer
  • Elution buffer
  • Spin columns
  • Collection tubes
  • Microcentrifuge

Procedure

  1. Tissue Disruption and Lysis:
    • Begin by disrupting the animal tissue sample. This can be achieved through mechanical methods like homogenization or by using a mortar and pestle.
    • Add lysis buffer containing detergents and enzymes (like Proteinase K) to the disrupted tissue. The lysis buffer breaks down cell membranes and nuclear envelopes, releasing DNA into the solution.
    • Proteinase K further digests proteins associated with the DNA.
    • Incubate the sample at an appropriate temperature to allow for complete lysis.
  2. Removal of RNA and Cellular Debris:
  1. Add RNase A to the lysate to degrade RNA, which can interfere with downstream applications.
  2. Centrifuge the sample to pellet cellular debris and other contaminants.
  3. DNA Binding to Spin Column:
  1. Transfer the supernatant containing DNA to a spin column.
  2. Add binding buffer to the column. The high salt concentration in the binding buffer promotes DNA binding to the silica membrane within the spin column.
  3. Centrifuge the column to bind DNA to the membrane and remove impurities.
  4. Washing:
  1. Wash the spin column with wash buffer to remove any remaining contaminants.
  2. Centrifuge the column to remove the wash buffer.
  3. DNA Elution:
  1. Add elution buffer (low salt buffer or water) to the spin column.
  2. Centrifuge the column to elute the purified DNA into a collection tube.

Tips and Considerations

  • Ensure all reagents and equipment are RNase-free to prevent RNA contamination.
  • Optimize lysis conditions (incubation time and temperature) for the specific animal tissue used.
  • Carefully follow the manufacturer’s instructions for the spin column kit.
  • Use appropriate safety measures when handling animal tissue and chemicals.

Applications of Extracted DNA

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

  • PCR (Polymerase Chain Reaction)
  • Genotyping
  • DNA sequencing
  • Gene expression analysis
  • Cloning

Conclusion

Spin column DNA extraction from animal tissue is a reliable and efficient technique widely used in research laboratories. By following the outlined steps and considering the provided tips, you can successfully isolate high-quality DNA for your molecular biology experiments.

Tissue Homogenize Buffer :

Tris-Hcl (pH)-8.0 : 1M

EDTA (pH. 8.0) : 0.5M

NaCl : 5M

SDS :20%

Lysis Buffer :

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

Binding buffer :

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 )

Protocol :

  • Cut up to 50-100 mg tissue into small pieces, and place in a 2.0 ml micro centrifuge tube Or pestle it into powders in liquid nitrogen, transfer 20-40mg powder into a 1.5ml tube. Add 180 μl Tissue homogenize Buffer  and mix well. 

  • Note: We strongly recommend to cut the tissue into small pieces to enable more efficient lysis. If desired, lysis time can be reduced by grinding the sample in liquid nitrogen before addition of Lysis Buffer and proteinase K.
  • Add 20 μl Proteinase K and 5 μl RNAse A. Mix thoroughly by vortexing, and incubate at 60°C until the tissue is completely lysed. Briefly invert the tube during incubation.
  • Note: Approximate 120-180 minutes depend on sample source. Check tissue lysis during the incubation. If tissue is completely lysed go for further step, higher incubation time after lysis of tissue may cause degradation of the DNA.
  • Add 200μl of Lysis buffer and Mix thoroughly by vortexing.
  • Incubate at 65°C for 10 minutes either on thermo mixer or water bath or dry bath. Briefly invert the tube once during incubation.

  • After incubation centrifuge entire lysate centrifuge 5000 rpm for 1 minute. Carefully aspirate supernatant without any debris and transfer into new 2.0 ml tube.

  • Add 300μl 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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