Dna Extraction

Topics: Cell, Protein, Solvent Pages: 3 (911 words) Published: October 12, 2012
EDTA tubes: tubes with purple colored caps; called so because they contain EDTA {Ethylene Diamine Tetraacetic acid} * Empty blood sample into falcon tubes-larger tubes that are graduated so lessen the work and can accommodate up to 50mL * Add a solution to dilute to 40 mL without touching to avoid cross contamination * Shake well

* Centrifuge for 10 minutes
DNA extraction takes place from the nuclei of leukocytes.
EDTA is simply an anticoagulant {otherwise if blood clots; DNA extraction will not be possible}. We rely on EDTA because it doesn’t interfere with later analysis such as diagnostic tests or molecular research. In this experiment we follow a series of steps in order to extract the DNA from the nucleus; this is done by: * Cell lyses using hypotonic solution. Cell lyses solution: mixture of salts whose tonicity is regulated such that it causes rupture of the cell membrane only. It is important to regulate the tonicity because initially; we don’t want to rupture the nuclear membrane. When we rupture the cell membranes {of WBCs}; the nuclei will be released, which can then be collected by centrifugation. * Next step to reach the DNA; is rupturing the nuclear membrane; same way we use nuclei lyses solution. Upon rupturing the nuclear membrane, DNA + RNA + proteins will be released. RNA will not cause an issue because it is a very sensitive molecule that will not be able to resist harsh conditions, and is easily degraded. The only problem is with the proteins. * In order to remove proteins; we make use of organic solvents: Phenol Chloroform – which should be used in the ratio 1 : 1 {for eg. If my blood sample is 10 mL; I use 5mL Phenol and 5mL Chloroform}. Phenol is very powerful in disrupting/degrading proteins and chloroform is a very powerful organic solvent. Two phases will be created : * Aqueous phase containing DNA

* Organic Phase
Degraded proteins will assemble at the boundary between the two phases....
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