DNA Isolation of Sugarcane via DNA Sequencing Process

      As part of our laboratory activity at IPB University, we conducted DNA isolation from sugarcane leaves—a fundamental step in DNA sequencing and plant molecular research. This procedure allowed us to extract high-quality DNA, which can later be used for various genetic analyses and applications in plant breeding and biotechnology.


      The process began with the lysis stage, where we disrupted the plant cells to release the DNA. Approximately 2 grams of sugarcane leaves were weighed, sprayed with alcohol to sterilize, and wiped with tissue. The leaves were then ground using a mortar and pestle along with CTAB extraction buffer, which helps break down cell walls and membranes. We added 700 µL of polyvinylpyrrolidone (PVP) to bind phenolic compounds and prevent DNA degradation. The mixture was transferred into 2 mL tubes, vortexed for one minute, and then incubated in a water bath at 65°C for 30 minutes, with the tubes inverted every 10 minutes to ensure proper mixing and reaction.


     Following cell lysis, we proceeded to deproteinization, which removes proteins and other unwanted materials. Chloroform:isoamyl alcohol (CI) was added to each tube up to 1.5 mL, and the tubes were gently inverted and centrifuged for 15 minutes. After centrifugation, the supernatant—the layer containing DNA—was carefully transferred to a new tube, while the pellet containing debris was discarded.


     In the precipitation stage, cold isopropanol was added to the supernatant until the tube was full. The samples were stored in a freezer for 1 to 24 hours to allow the DNA to precipitate. After freezing, they were centrifuged for 40 minutes, the isopropanol was discarded, and the resulting DNA pellet was left to dry.


    Lastly, we performed purification to clean the DNA. Cold absolute ethanol (300 µL) was added, followed by another 15-minute centrifugation. The ethanol was removed, and the pellet was dried again. Finally, the purified DNA was dissolved in 100 µL of double-distilled water (ddH₂O), making it ready for further analysis.



      This hands-on activity gave us a deeper understanding of how DNA is extracted and prepared for sequencing. It was fascinating to see how each step—lysis, deproteinization, precipitation, and purification—played a critical role in isolating usable DNA. The experience also highlighted the importance of DNA technology in advancing agriculture, particularly in crop improvement and genetic research. Overall, it was a rewarding and educational experience that connected theoretical knowledge with real-world laboratory techniques.

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