To obtain DNA fragment with desired gene, the following steps have to be taken :
1. Opening the cell either by each of the method below :
Enzymatic method : - enzymatic digestion and cell membrane lysis
- this method created "holes" to release DNA from cells
- example : lysozyme is used to break the cell wall of bacteria
Physical method : - mechanical shearing by using centrifugtion
- grinding the organisms by using pestle and mortar if it is a plant
- this method created "holes" to release DNA from cells
- example : lysozyme is used to break the cell wall of bacteria
Physical method : - mechanical shearing by using centrifugtion
- grinding the organisms by using pestle and mortar if it is a plant
2. Deproteinization using phenol : chloroform In this step, protein will be eliminated due to different solubility among DNA, RNA, protein and other constituent in the cells. Upper aqueous phase : DNA and RNA Lower phase : unwanted constituent Interphase : protein The process repeat several times until interphase is clear. The right top picture is a schematic diagram that explain on the dissolution of different cellular component in different solution. The right bottom, picture shows the real diagram of phenol:chloroform deproteinzation. |
3. Purification of DNA
After protein and cell debris was removed from the solution, the next step is to eliminate RNA from the solution. Since both DNA and RNA are dissolve in the aqueous layer of phenol:chloroform extraction method, RNA can only be eliminate by using the enzyme that digest RNA, that is RNase. |
4. Polymerase Chain Reaction (PCR)
Sometimes, the DNA that extracted is in very low amount. Therefore, the extracted DNA have to undergo PCR to amplify the amount of DNA that available for the subsequent steps in gene cloning. |
5. DNA Quantification
Quantification is an important step to measure the quantity and purity of DNA that we extract. The DNA extracted are measured by UV absorbance spectrometry at the wavelength of 260nm. In practical, the A260 value of spectrophotometry provides a measure of concentration:
It can also check for DNA purity : The A260/A280 ratio provides an estimate of purity:
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6. Gel Electrophoresis After the DNA was amplify, the DNA with specific sequence will be identify by gel electrophoresis. The DNA sequence will be intercalated by ethidium bromide (EtBr) to visualized under radiography. Gel electrophoresis will separate desired sepcific sequence from bunches of DNA by separating them using electric field according to size of DNA. Since DNA is negatively charged, they will move towards the positive electrode of gel electrophoresis. The separated DNA can then be ressolved from gel for subsequent steps. |