The extraction of DNA from plant leaves or seeds is critical to the development of future plant-based technologies. Many downstream molecular biology techniques, such as PCR and NGS, rely on plant DNA, and nucleic acid extractions are central to the workflows of many academic researchers and commercial breeding programmes. DNA is being used in broader studies to refine and redefine our understanding of plant evolution and adaptation, while also providing information for conservation, crop breeding, and food security.
Here’s a guide to extracting DNA from plants.
In general, plant DNA extraction procedures must include the five steps listed below:
Cell wall breakdown: To break down the cell walls and release the cellular contents, grind the tissue in dry ice or liquid nitrogen with a mortar and pestle.
Cell membrane breakdown: The cell membranes must also be disrupted after the cell wall is broken in order for the DNA to be released into the extraction buffer. This is accomplished by using a detergent, typically CTAB or SDS buffers.
DNA protection: Endogenous nucleases can degrade DNA once it is released into the extraction buffer. Some reagents, such as EDTA (ethylenediaminetetraacetic acid), are used to prevent DNA degradation. EDTA is a chelating agent that binds magnesium ions, which are required cofactors for most nucleases.
DNA isolation: Using a Plant DNA Isolation Kit, the DNA in the extraction buffer is separated from other components (such as proteins, polysaccharides, and phenolics). To achieve isolation, the buffer/tissue mixture is emulsified with either chloroform or phenol to denature and separate the contaminants from the DNA.
DNA precipitation: Following the removal of contaminants, the DNA is precipitated with ethanol (generally 70%) and later dissolved in water. Finally, the DNA is kept at -20°C.
Before you begin, create a written protocol. It is critical to have a written protocol in place before beginning the extraction, and to double-check the steps as you go. It will allow you to check for errors if the desired results are not obtained. It also provides an opportunity for improvement because changes or deviations can be easily noted on the protocol and compared to better or worse yields.
Prepare all materials ahead of time. All DNA protocols call for sterilized and cleaned material that is free of DNases and RNases. It usually takes time to prepare. So, sterilize the mortar and pestle ahead of time, and have aseptic tips, falcon tubes, and microcentrifuge tubes on hand.
Set a time for DNA extraction. Allow enough time for DNA extraction when optimizing a protocol. Some protocols may necessitate cleaning rounds or overnight incubations. Starting early is recommended because you may encounter issues that are difficult to resolve after work hours.
Always review after failing. Don’t give up after a failed DNA extraction. It is natural to be frustrated after numerous failed attempts. Follow each step of your DNA extraction protocol carefully, and if you followed our first tip, you should be able to easily go back and determine the likely cause of the error.
To buy a DNA Isolation Kit in Tucker, GA, get in touch with us at AMD Biotech.