DNA extraction is a fundamental technique in molecular biology, with applications ranging from medical diagnostics to food safety testing. While DNA is most commonly associated with blood samples, it can also be extracted from other sources, including milk.
Milk contains somatic cells, which are body cells that are naturally shed from the mammary gland during milk production. These cells contain DNA, which can be extracted and analyzed. One efficient method for DNA extraction from milk is using a silica column.
What is a Silica Column?
A silica column is a small tube filled with silica beads. Silica has a high affinity for DNA, meaning that DNA molecules will bind to the silica beads under specific conditions. This allows for the separation of DNA from other components in the milk sample.
The Process:
- Milk Sample Preparation: A small volume of milk is typically used for DNA extraction. The milk sample may be treated with enzymes or chemicals to break down proteins and other cellular components, making the DNA more accessible.
- Lysis: A lysis buffer is added to the milk sample to break open the somatic cells and release the DNA.
- Binding: The lysate is then passed through the silica column. Under specific conditions, the DNA will bind to the silica beads, while other components will pass through the column.
- Washing: The column is washed with buffers to remove any remaining impurities.
- Elution: Finally, a low-salt buffer or water is used to elute the DNA from the silica column. The eluted DNA is now ready for downstream applications, such as PCR or sequencing.
Advantages of Using a Silica Column:
- Efficiency: Silica columns provide a high yield of pure DNA.
- Simplicity: The process is relatively straightforward and can be performed with basic laboratory equipment.
- Scalability: The method can be easily scaled up or down depending on the volume of milk and the desired amount of DNA.
Applications:
DNA extracted from milk can be used for various purposes, including:
- Breed Identification: DNA analysis can be used to identify the breed of a cow, which is important for dairy herd management and breeding programs.
- Disease Diagnosis: DNA extracted from milk can be used to diagnose diseases in cows, such as mastitis or bovine leukemia virus.
- Food Safety Testing: DNA analysis can be used to detect the presence of pathogens or adulterants in milk.
Conclusion:
DNA extraction from milk using a silica column is a valuable tool for researchers and industry professionals. Its efficiency, simplicity, and scalability make it a preferred method for many applications. As DNA analysis continues to advance, we can expect to see even more innovative uses for this technique in the future.
TE Buffer Ingredients:
- 10 mM Tris-HCl
- 1 mM EDTA
Lysis Buffer :
Tris-Hcl (pH)-8.0 : 1M
EDTA (pH. 8.0) : 0.5M
NaCl : 5M
SDS :20%
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 ,Tris-HCl and Triton X-100
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 :
- Transfer upto 2 ml milk sample into a microfuge tube (provided by user). Centrifuge at full speed (13,000 rpm) for 2 min .
- Discard medium completely and re-suspend the pellet in 180 μl TE Buffer or Elution Buffer. Centrifuge at full speed (5000 rpm) for 5 min and discard the supernatant
- Add 200μl of Lysis buffer and 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 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 new collection tube. Add 500μl of binding buffer . Centrifuge at 12,000 rpm for 2 min. discard the flow through.
- Place the column into new 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 new 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) (In this tube DNA will be eluted in 1.5 ml eppendrof tube so don’t discard it ).
- Centrifuge 10,000 rpm for 1 min to elute pure 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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