Isolating DNA from insects is a common procedure in various biological research areas, including taxonomy, ecology, and genetics. The spin column method offers a rapid and efficient way to obtain high-quality DNA from insect samples. This blog post will provide a general overview of the DNA extraction process from insects using a spin column.
Materials Required
- Insect 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:
- Depending on the size of the insect, you may need to homogenize or grind the sample using a pestle or homogenizer.
- Add lysis buffer to the insect sample. 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 fresh or properly preserved insect samples for optimal DNA yield and quality.
- Ensure all reagents and equipment are RNase-free to prevent RNA contamination.
- Optimize lysis conditions (incubation time and temperature) for the specific insect species and sample type.
- Carefully follow the manufacturer’s instructions for the spin column kit.
Applications of Extracted Insect DNA
The DNA extracted using the spin column method can be used for various applications, including:
- Species identification and barcoding
- Population genetics studies
- Phylogenetic analysis
- Forensic entomology
- Insect pest management
- Gene expression analysis
Conclusion
Spin column-based DNA extraction from insects is a widely used and reliable technique in entomological research and other fields. By following the outlined steps and considering the provided tips, you can successfully isolate high-quality insect DNA for your downstream applications.
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 )
Extraction Protocol :
Homogenise up to 100 -125 mg insects in 600 μl of Homogenise buffer (Ice Chilled) using a mortar and pestle. Transfer the entire homogenise in a 2.0 ml microcentrifuge tube.
Add 20 μl of Proteinase K and 20 μl of RNAse A to the microcentrifuge tube .
Add 500 μl of Lysis Buffer and Mix thoroughly by vortexing for 30 seconds .
Incubate at 65°C for 30 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. 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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