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Abstract
Trichoderma fungi cell walls are composed of chitin compounds that are very sturdy and resistant to enzyme activity. The specific techniques and methods are needed to degradate of fungi cell wall. This study aims to analyze the best DNA isolation method in lysing fungal cell walls by comparing several isolation methods. The methods are using DNeasy Plant Mini Kit, DNeasy Plant Mini Kit combination with heating, and DNeasy Plant Mini Kit combination in a physical way (grinding the sample using mortar and pestle in liquid nitrogen). This research was conducted in March to September 2018 at the Microbiology Laboratory, Laboratory of Biotechnology and Genetics, as well as the Integrated Research Laboratory, FMIPA, UNP. Data were analyzed qualitatively by agarose gel electrophoresis and quantitatively by calculating the purity and concentration of DNA using Nanodrop. The results showed that the DNeasy Plant Mini Kit combination method by physical means gave the highest concentration of DNA isolation of 2.4 μg / ml with a purity value of 1.857. In the DNeasy Plant Mini Kit method and the DNeasy Plant Mini Kit combination method with heating, the DNA concentrations obtained were 1.211 µg / ml and 0.933 µg / ml respectively with a purity value of 1.728 and 1.708. The results of the electrophoresis test showed a thin band of DNA bands in samples isolated by a combination of kit methods and physical methods, whereas in the other two samples no DNA bands were found. It can be concluded that the method of DNA isolation that can be used as a reference to degrade Trichoderma mushroom cell walls is a combination of kit and liquid nitrogen method with a note that additional levels of isolates are needed.
Highlights
This study aims to analyze the best DNA isolation method in lysing fungal cell walls by comparing several isolation methods
The results showed that the DNeasy Plant Mini Kit combination method by physical means gave the highest concentration of DNA isolation of 2.4 μg / ml with a purity value of 1.857
The results of the electrophoresis test showed a thin band of DNA bands in samples isolated by a combination of kit methods and physical methods, whereas in the other two samples no DNA bands were found
Summary
Tujuan dari penelitian ini adalah untuk menganalisis metode isolasi DNA yang terbaik dalam melisiskan dinding sel jamur. 0,1 gram, lalu dimasukkan ke dalam tabung eppendorf volume 1,5 ml yang berisi 400 μl buffer AP 1, lalu suspensi divorteks sampai homogeny dan ditambahkan RNAse 4 μl, kemudian dipanaskan lagi pada suhu 65oC selama 10 menit sambil dibolak balik 2 sampai 3 kali. Sebanyak 650 μl campuran dipindahkan ke dalam mini spin column, kemudian disentrifugasi selama 1 menit dengan kecepatan 8000 rpm, setelah itu cairan yang ada pada tabung dibuang. Buffer AE ditambahkan sebanyak 100 μl, lalu diinkubasi selama 5 menit pada suhu ruang, kemudian sentrifugasi lagi 1 menit dengan kecepatan 8000 rpm. 2.2.3 Isolasi DNA dengan Kombinasi Metode DNeasy Plant Mini Kit dan Pemanasan Isolasi DNA dilakukan dengan cara jamur yang berumur 4 hari diambil sebanyak. Visualisasi dan analisis DNA dilakukan di UV Transilluminator dan hasilnya difoto dengan menggunakan kamera hp Xiomi model MI 4W
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