Chapter 13 :DNA Extraction From Mouse Tail

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DNA extraction from mouse tails is a common practice in research labs for genotyping and other genetic analyses. The spin column method offers a fast and convenient way to isolate high-quality DNA from this tissue source. This blog post will outline the general procedure for DNA extraction from mouse tails using a spin column.

Materials Required

  • Mouse tail sample
  • Lysis buffer
  • Proteinase K
  • RNase A
  • Binding buffer
  • Wash buffer
  • Elution buffer
  • Spin columns
  • Collection tubes
  • Microcentrifuge
  • Pestle or homogenizer

Procedure

  • Sample Preparation and Lysis:
    • The mouse tail sample is typically digested in a lysis buffer containing Proteinase K. This buffer breaks down cell membranes and proteins, releasing DNA into the solution.
  • RNA Removal:
    • RNase A is added to the lysate to degrade RNA, which can interfere with downstream applications.
  • DNA Binding to Spin Column:
    • Binding buffer is added to the lysate to create conditions that promote DNA binding to the silica membrane within the spin column.
    • The lysate is then transferred to the spin column.
    • Centrifugation of the column allows the DNA to bind to the membrane while impurities are removed.
  • Washing:
    • The spin column is washed with a wash buffer to remove any remaining contaminants.
    • Centrifugation is again used to remove the wash buffer.
  • DNA Elution:
    • An elution buffer (low salt buffer or water) is added to the spin column.
    • A final centrifugation step elutes 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 efficient tissue digestion and DNA release.
  • Handle mouse tail samples with care and follow appropriate safety guidelines.
  • Carefully follow the manufacturer’s instructions for the specific spin column kit being used.

Applications of Extracted Mouse Tail DNA

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

  • Genotyping
  • Transgenic mouse identification
  • Gene expression analysis
  • DNA sequencing

Conclusion

Spin column-based DNA extraction from mouse tails is a routine and essential technique in many research settings. By following these steps and considering the provided tips, researchers can efficiently obtain high-quality DNA for their experiments.

Homogenize Buffer :

Tris-Hcl (pH)-8.0 : 1M

EDTA (pH. 8.0) : 0.5M

NaCl : 5M

SDS :20%

Lysis 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.

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 0.2–0.5 cm (25-50 mg) mouse tail into small pieces, and place in a 1.5ml microcentrifuge tube. Or pestle it into powders in liquid nitrogen, transfer 20-40mg power into a 1.5ml tube. Add 180 μl Homogenize buffer and mix well


  • 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 add 100μl 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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