I. TITLE: isolation of DNA
II. PURPOSE:
· To find a simple method to isolate DNA fruit.
· Knowing the effectiveness of detergent/soap use for DNA isolation.
III. BASIC THEORY:
DNA (Deoxiribose Nucleic Acid) is composed of three main components of the sugar deoxyribose, nitrogenous bases and phosphate to form nucleotides incorporated. (Istanti,1999). The DNA molecule is bound to form chromosomes, and is found in the nucleus, mitochondria and chloroplasts. DNA that make up this chromosome is composed of nucleotide double helix double (double helix), in which the nitrogen bases and the second string in each pair polynucleotide pairs through hydrogen bonds and fixed between a single nucleotide with another nucleotide associated with the phosphate bond. DNA contained in every living cell and called, the blueprint of life, because this molecule plays an important role as a carrier of hereditary information that determines the structure of proteins and other metabolic processes. (Tim Dosen Genetika)
To obtain pure DNA from a cell in the body tissues of living things can be done a DNA isolation technique. Isolation of DNA can be done in various ways, but in any type or plant parts can cause different problems, among others because of polyphenolic compounds and polysaccharides in high concentrations can inhibit DNA purification and also affect the enzyme- enzymes such as polymerase, ligase, restriction endonuclease, or other enzymes for molecular events that can lead to DNA can not be used for research applications. (Fried, 1992).
DNA is composed of three main components ie deoxyribose sugars, nitrogenous bases, and phosphate tergabungmembentuk nucleotides. DNA contained in every living cell a very important role as a carrier of hereditary information that determines the structure of proteins and other metabolic processes. isolation of DNA can be done through the stages include: preparation of cell extracts, purification of DNA from ekstrsk cells and DNA precipitation. Although the isolation of DNA can be done in various ways, but in any kind or parts of plants may give different results, this is due to polyphenolic compounds and polysaccharides in high concentrations can inhibit DNA purification. If the isolation of DNA carried out with samples of fruit, then the water content of each fruit is different, can give different results too. The higher the water content, the cells are dissolved in the extract will be less, so that DNA terpretisipasi will also be small. (Istanti, 1999).
The addition of detergent in the isolation of DNA can cause damage to cell membranes, through the bonds formed through the side of the hydrophobic detergent with protein and fat in the membrane forming compounds' lipid protein-detergent complexes ". This compound can be formed by proteins and lipids have hydrophilic and hydrophobic ends, as well as the detergent, so it can form a chemical bond.
* DNA also can be isolated, both in humans and in plants. Human DNA can be isolated through the blood. Human blood consists of blood plasma, globulus fat, chemical substances (carbohydrates, proteins and hormones), and gases (oxygen, nitrogen and carbon dioxide). Blood plasma consists of erythrocytes (red blood cells), leukocytes (white blood cells) and thrombocytes (platelets). Blood components were isolated white blood cells. White blood cells be selected as a nucleus, where there is DNA in it. DNA in plants can also be isolated, for example in plants of onion (Allium cepa) and the banana (Musa sp.) (Kimball, 1992).
* Isolation of DNA has several stages, namely:
oIsolation of tissue
oWall and cell membrane dilisiskan
oExtracted in a solution
oPurified
oPrecipitated
The principles in conducting DNA isolation there are two, namely centrifugation and precipitation. The main principle is to separate substances by centrifugation specific gravity of the molecule by providing a centrifugal force so that the heavier substance will be at the bottom, while the lighter substance will be located at the top. Centrifugation technique was performed in a machine called the centrifuge machine with variable speed, eg 2500 rpm (rotation per minute) or 3000 rpm (Hays Lana, 2005).
There are 5 stages to perform DNA isolation, namely: network isolation, pelisisan cell walls and membranes, extracting in solution, purification, and precipitation.
The first stage is carried out to isolate the network you want to use, namely blood.
The next stage which lyse the cell walls and membranes with a solution of red blood cells pelisis. After incubation, the blood that has been mixed with pelisis red blood cells are then centrifuged for 10 minutes at 2500 rpm. Furthermore, the supernatant was removed and later formed in the extraction solution. It is intended that the nuclear extracts derived white blood cells.
The next stage is purification. This stage aims to clean the white blood cells of other substances, and the last stage, which aims to precipitate precipitation histone proteins, so that DNA strands are no longer roll up (coiling) and binds to histone proteins, which cause DNA to become visible (Kimball 2005).
Network isolation stage; to isolate the white blood cell tissue, the blood that still have complete components need to be separated from one another so that the only remaining white blood cells. Because it into a tube containing a solution of blood given pelisis red blood cells which is the solution hipotonis. Because the solution hipotonis, it will happen hemolysis. Pelisis red blood cell solution consisting of EDTA (ethylenediamine tetraacetic acid) which will form a complex (chelate) with metal ions such as Mg 2 + which is a cofactor DNase. Furthermore, the tube inverted denan circular movements that make up the number 8 in order to blend with the perfect solution for 10 minutes. Blood that has been mixed with the red blood cell pelisis then centrifuged for 10 minutes at 2500 rpm. Furthermore, the supernatant discarded formed. To lyse the cell membrane and nuclear membrane of white blood cells isolated before, given white blood cell pelisis solution consisting of EDTA and SDS (sodium dodecyl sulfate), which serves to damage the lipids in cell membranes so that the destroyed leukocytes.
The next stage of purification. Purification aims to clean the white blood cells of other substances; To the solution was then administered RNAse and incubated for 15 min at 37 ° C. This work aims to optimize the enzyme that is strongly influenced by temperature. The next stage of the precipitation; Phase precipitation is done by dripping a solution of protein precipitation and then divortex that aim to homogenize the solution. Protein precipitation solution consisting of ammonium acetate which, when bound to the protein resulted in the formation of new compounds that have a lower solubility, thus causing the protein precipitate. The solution is then centrifuged again for 15 minutes at 3000 rpm. Supernatant containing the DNA is then poured into a tube containing cold isopropanol and the tubes inverted back with a figure 8 motion. Provision of isopropanol aims to visualize the DNA. Furthermore, the tubes were centrifuged again for 5 minutes at 3000 rpm. The results of the centrifugation pellet is the presence of DNA on the basis of the tube which was then added 70% ethanol and inverted again. Giving ethanol aims to clean up the DNA of the impurity-pengotornya. Once mixed, the tubes and then centrifuged again for 5 minutes at 3000 rpm. The end result is a DNA that is on the edge of the bottom of the tube. Last step is the provision of Tris-EDTA which aims to re-dissolve the DNA for preservation (Hays Lana, 2005).
IV. OBSERVATION METHOD
4.1 Tools and Materials
1. Beaker glass or glass aqua
2. knife
3. mixer
4. Filters (tissue / cotton)
5. engine blender
6. Spatula
7. Reaction tube and tube rack
8. Tomatoes, papaya, pears
9. Detergents (Surf, Rinso)
10. Soap dab (Bukrim)
11. salt
12. Cold 96% ethanol (ethanol and ice cubes)
13. aquades
4.2 How to WorkV.
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VIII.
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V. OBSERVATION RESULT
| No | Mixture | Before droping etanol | After droping etanol | Occuration DNA |
| 1 | Tomato + Rinso (2,3 ml) Etanol (1,92 ml) | One layer Yellow muddy collored | 3 layer (from bottom) 1. Tomato 2. DNA 3. Rinso + etanol | 113 second |
| 2 | Pear + Rinso (2,1ml) Etanol (1,75 ml) | One layer Transparant And soft yellow color | 4 layer (from bottom) 1. Pear essense 2. Etanol 3. DNA 4. Detergen | 46 second |
| 3 | Papaya + Rinso (2,6ml) Etanol (2,16 ml) | The color is clearly orange Consist of one layer | 3 layer (from upon) 1. Rinso + etanol 2. DNA 3. Fruit essense and another part of papaya juice | 76 second |
| 4 | Tomato + Surf (3,5ml) Etanol (2,9ml) | The color is yellow clear Only one layer | 3 layer (from bottom) 1. Detergen+aquades 2. DNA 3. Tomato+etanol | 54 second |
| 5 | Pear + Surf (2,7ml) Etanol (2,5ml) | The color is yellow clear, consist of one layer | 3 layer (from bottom) 1. Surf+etanol 2. DNA 3. Pear essense | 68 second |
| 6 | Papaya + Surf (2,2ml) Etanol (1,83ml) | The color is orange pale There are one layer | 3 layer (from bottom) 1. Surf + etanol 2. DNA 3. Papaya essense | 85 second |
VI. ANALYSIS
Percobaan kali ini berjudul isolasi DNA. Isolasi DNA yang dilakukan pada percobaan ini adalah isolasi DNA pada buah, yaitu buah pir, pepaya, dan tomat. Praktikum kali ini bertujuan to find a simple method to isolate DNA fruit and knowing the effectiveness of detergent/soap use for DNA isolation.
Langkah pertama yang dilakukan dalam percobaan ini adalah menimbang bahan (buah pir, pepaya, dan tomat) sebanyak 100 gram kemudian diblender dengan aquades 100 ml selama 40 detik atau sampai bahan hancur. Tujuan dari proses pemblenderan ini adalah untuk mengeluarkan DNA dari dalam sel. Hal ini dapat dilakukan dengan merusak dinding sel, membran sel, dan membran inti.. Setelah diblender kemudian 4ml hasil blender atau jus dicampur dengan 4 ml larutan detergen tertentu, detergen yang digunakan adalah rinso dan surf. Lalu tambahkan 1 spatula garam dapur dan aduk selama 10 menit. Setelah diasuk kemudian disaring sebanyak dua kali. Pada hasil penyaringan kedua ditambahkan etanol yang banyaknya sudah disesuaikan dengan banyaknya hasil penyaringan. Kemudian mencatat waktu yang diperlukan sampai terlihat endapan dan perubahan warna yang terjadi.
Detergen yang digunakan berfungsi untuk melisiskan barier (penghalang) sel secara kimia sebagai pengganti senyawa kimia yang mampu merusak dinding dan membran sel antara lain lisozim yang dapat mendegesti senyawa polimerik yang menyebabkan kekakuan sel dan etil endiamintetra asetat (EDTA) yang berfungsi untuk menghilangkan ion Mg2+ yang penting untuk mempertahankan keseluruhan struktur selubung sel, serta menghambat enzim-enzim seluler yang dapat merusak DNA (ion Mg2+ merupakan kofaktor penting bagi DNAse yang biasa "memakan" DNA). Deterjen dapat menyebabkan rusaknya mebran sel, melalui ikatan yang dibentuk melalui sisi hidrofobik deterjen dengan protein dan lemak pada membran membentuk senyawa ”lipid protein-deterjen kompleks”. (Hays Lana, 2005).
Garam digunakan untuk melarutkan DNA, karena ion Na+ yang dikandung oleh garam mampu memblokir (membentuk ikatan) dengan kutub negatif fosfat DNA, yaitu kutub yang bisa menyebabkan molekul-molekul saling tolak mnolak satu sama lain sehinggga pada saat ion Na+ membentuk ikatan dengan kutub negatif fosfat DNA, DNA akan terkumpul (Dollard, 1994, dalam Jamilah, 2005: 21). Dari pernyataan tersebut, nampak bahwa selain digunakan untuk menghilangkan protein dan karbohidrat dan menjaga kesetabilan pH lysing buffer, garam juga membantu proses pemekatan DNA. (Hays Lana, 2005).
Pemberian Alcohol dingin (etanol) pada percobaan ini betujuan untuk melakukan presipitasi sehingga DNA yang telah terkumpul tadi mampu memisah dari larutan dan terbentuklah lapisan-lapisan yang dapat diidentifikasi unsur penyusunnya. DNA dapat mengalami denaturasi dan renaturasi. (Hays Lana, 2005).
Dari data yang diperoleh dapat dilihat bahwa macam detergen dan tingkat kadar air yang terkandung dalam buah mempengaruhi jumlah dan kualitas DNA yang muncul dalam isolasi DNA, selain itu juga mempengaruhi waktu yang dibutuhkan essence untuk mengendap dan menghasilkan DNA. Data menunjukkan bahwa macam detergen mempunyai pengaruh berbeda pada jenis buah tertentu.
Pada percobaan group 1 mixture tomato and rinso 2,3 ml dengan etanol 1,92 ml. Pada penyaringan kedua sebelum ditetesi etanol hanya terdapat 1 layer dan have yellow muddy collored. After dropping by etanol there are consist 3 layer after 113 second. While, in the same mixture in group 4, but make different detergen that is Surf. Sebelum ditetesi dengan etanol hanya ada 1 layer dengan yellow clear or transparant colour. After dropping by etanol there are consist 3 layer after 54 second. Lebih cepat 59 detik daripada percobaan grup 1 yang menggunakan detergen rinso.
Pada percobaan group 2 mixture pear and rinso 2,1 ml dengan etanol 1,75 ml. Pada penyaringan kedua sebelum ditetesi etanol terdapat 1 layer dan have transparant and soft yellow colour. After dropping by etanol there are consist 4 layer after 46 second. While, in the same mixture in group 5, but make different detergen that is Surf. Sebelum ditetesi dengan etanol hanya ada 1 layer dengan transparant and soft yellow colour. After dropping by etanol there are consist 3 layer after 68 second. Lebih lambat 22 detik daripada percobaan grup 1 yang menggunakan detergen rinso.
Pada percobaan group 3 mixture papaya and rinso 3,5 ml dengan etanol 2,91 ml. Pada penyaringan kedua sebelum ditetesi etanol terdapat 1 layer dan have clearly orange or transparant colour. After dropping by etanol there are consist 3 layer after 76 second. While, in the same mixture in group 6, but make different detergen that is Surf. Sebelum ditetesi dengan etanol hanya ada 1 layer dengan orange pale colour. After dropping by etanol there are consist 3 layer after 85 second. Lebih lambat 9 detik daripada percobaan grup 1 yang menggunakan detergen rinso.
Detergen yang digunakan pada percobaan ini memberikan pengaruh yang berbeda. Seperti yang disebutkan di atas bahwa detergen yang digunakan adalah rinso dan surf. Secara keseluruhan rinso memberikan effect reaksi yang lebih cepat kecuali pada percobaan mixture with tomato. Cepatnya reaksi dalam hal ini ditandai dengan cepatnya waktu yang dibutuhkan untuk tercipta endapan dan pemisahan layer. Detergen surf memberikan effect reaksi yang lebih lambat dibandingkan detergen rinso.
Jenis buah dan jenis detergen berpengaruh terhadap hasil isolsi DNA. DNA pada masing-masing buah mempunyai kadar atau jumlah yang berbeda dan warnanya pun berbeda. Jenis detergen juga berpengaruh terhadap kecepatan pembentukan DNA
VII. CONCLUSION:
1. DNA isolation technique, is a way / method to separate DNA from cells, either from the nucleus, mitochondria and chloroplasts. Isolation of DNA can be done through the stages include: preparation of cell extracts, purification of DNA from cell extracts and DNA precipitation.
2. Types of detergents affect the results of DNA isolation on the fruit, the level of quality detergents in order to form the DNA is varied depending on the Janis fruit and fruit water content. The best detergent used is surf detergent.
3. REFERENCES
Fried, G.H & Hadenemas. 1992. Biologi. Jakarta: Erlangga.
Hays, Lana. 2005. Introduction to DNA Extraction, (Online), (http://www.tsl.orst.edu.tgerc/dnaext.html, accessed 14 November 2011)
Istanti, Annie. 1999. Biologi Sel. Malang: jurusan Biologi FMIPA UM.
Kimball, john w. 1992. Biologi (jilid 5). Jakarta: Erlangga
Tim Dosen Genetika. 2011.Petunjuk Praktikum Genetika. Jember: University Press
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