Sodium dodecyl sulphate (SDS) which have been used in isolation the DNA of plant as reagent while in isolating the DNA of soils and microbes as a buffer is actually anionic detergent. The main function of this reagent and buffer are similar to CTAB which is to destroy lipid structure in the cell membrane. Thus, SDS is usually added in the step of lysis purposely to breakdown the cell and releasing the nucleic acid.
Other than buffers, there are also another some reagent that are used in order to extract DNA. Proteinase K which have been added after the process of lysis in isolation DNA of insect, invertebrate organisms, soils and microbes are aimed to digest the contamination of protein caused by the extraction of nucleid acid (DNA). Proteinase K also can helps to degrade nucleases which may have present that can cause to degradation of DNA. Chloroform have a feature in helping to solubilizes lipids together with proteins. In the context of DNA extraction, chloroform have a role in separating protein and polysaccharides from the nucleic acid by binding to them and as the chloroform have much denser than water, the content will separated into two distinct phase. Chloroform with bind organic matter will be at the lower phase while the upper will be the water contained DNA.
Usually, the used of chloroform in isolating DNA of insect and invertebrate animals are together with phenol and isoamyl alcohol (phenol:chloroform:isoamyl alcohol , 25:24:1), while in the DNA isolation of soils and microbes, chloroform will be mix with isoamyl alcohol (chloroform:isoamyl alcohol, 24:1). Isoamyl alcohol have a function to decrease bubbling and maintain the interphase between the aqueous and organic phase while phenol used to create a good interphase as it is slenderly soluble in water.But, in isolating the plant’s DNA the role of chloroform is taking over by the potassium acetate. Potassium acetate have a characteristic in making it less soluble in water by neutralize the negative charges of DNA. Thus, this result in separating two distinct phases which aqueous and organic phase.
Besides, potassium acetate also initiates the pelleting process.In general, the DNA is insoluble in the alcohol. For that reason, alcohol is used usually in the step of precipitation DNA. There are two types that are commonly used which are isopropanol and ethanol. Isopropanol used in most of DNA isolation have a function to precipitate the DNA at the wall of centrifuge tube while 70% of ethanol is used in DNA isolation of insects, invertebrate organisms and plants purposely to clean the DNA and remove the salt and other impurities that have been carry from the precipitation step.