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DNA out of the cells: DNA extraction
In molecular biology procedures, DNA extraction is a preliminary method. Methods for DNA extracting differ from plants to animals to bacteria. Even among bacteria there are different procedures involving DNA extraction. These variations are mostly due to the differences in their structure. For an instance, fungi have a thick wall consisting of chitin which is difficult to degrade. DNA extraction procedure should be monitored carefully as it is temperature sensitive and pH sensitive. It is essential to store extracted DNA samples at -20C. All the equipment used for DNA extraction should be sterilized. For microbial DNA extraction, it is important to use pure cultures. At the same time, using fresh samples is of significant importance.

In any method of DNA extraction, the wall or the membrane of the cell is broken using a detergent. After cellular constituents some out, nuclear membrane is lysed if present. The impurities like proteins and cell debris are removed by centrifugation. Isopropanol is added to precipitate proteins. Naked DNA is generally stored in a buffer solution at a temperature below freezing point. It is essential to prevent extracted DNA from degradation. If DNases are present in the stored extract, genetic material would be lysed.

In digestion of cell wall, content of lysis solution will vary depending on the organism. Plant cell walls contain cellulose. If high amount of cutin is present on plant material, digestion will be different from normal procedure. Extraction of DNA from plant materials usually involves crushing of plant material in liquid nitrogen. Chitin is the major constituent of fungal cell walls which makes it very tedious procedure. Enzymatic digestion is quite effective in lysing cell walls in case of microbes. Since it is costly various other chemical and physical methods are used. Chemicals used include detergents such as EDTA or SDS. These detergents can be ionic or non-ionic. The lysis solution usually contains a buffer to maintain pH and a salt. Physical methods include high temperature treatments, microwave treatments, high pressure applications, sonication etc. However both chemical and physical methods can lead to destruction of the genetic material. In case of DNA extraction from bacteria, lysis of the cell wall greatly depends upon whether the bacteria are gram negative or gram positive.
Apart from these, extracting DNA from different human cells such as red blood cells has specific procedures. If the specimen is pure, the purification is much easier. If it is associated with more contaminants, additional purification procedures may be necessary. For an instant, for microbial cultures its important the sample to be free of agar as it can inhibit Polymerase chain reaction, which is the next step in case of many molecular biology procedures.

After the cell wall is degraded, the nucleus will be exposed in case of eukaryotic organisms. Next the nucleus membrane will be lysed using a detergent. Nuclear lysis solution won’t have to be a strong one. In many organisms, plasma membrane structure is basically the same.
RNA content is important in DNA extraction. RNA can be removed by using RNase enzyme which shows its optimum activity at 370 C. RNA content is different in organisms. In case of Yeast and plants it’s generally high. Depending on this, the concentration of RNase can be increased to minimize RNA content in the final extract.
For protein removal, generally an ammonium salt is used which precipitates proteins with other cell debris. Protein removal is important as DNA of some organisms is associated with high content of proteins. This protein removal also removes the enzyme system of the cells. This is a crucial as enzymes such as DNase can lead to degradation of genetic material. A mixture of phenol and chloroform is used for protein purification in traditional methods.

After digestion of cell wall and membranes and digestion of protein and RNA, the retained cell debris can be removed by centrifugation. The speed of centrifugation for this is usually 10000 g. After centrifugation, the cell debris will make a precipitate at the bottom and the supernatant will contain DNA. When isopropanol is added to supernatant, DNA forms a thread like strand associated with air bubbles. Then the sample will be centrifuged to a higher speed to precipitate DNA followed by ethanol wash.

For storing DNA, Millipore water or TE buffer can be used. This should be neutral and free from contamination.
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