MadSci Network: Molecular Biology |
Hi Leah! The exact procedure and ingredients used for DNA extraction will depend on several factors, including what type of DNA you are extracting (genomic or plasmid), what is your source of DNA (tissue, bacteria, cell lines) and how pure you want the final preparation to be. Here are some general guidelines as to what the different steps are doing: 1. Firstly, you need to get the DNA out of the cells. To do that you must break the cells down. The best way to do this is to weaken the cells walls and then lyse them. If your DNA is in bacterial cells, you would probably use an enzyme like lysozyme to weaken them, and a combination of EDTA and a detergent to lyse them. For tissues, this might change to proteinase K and detergent. 2. The enzymes and detergent are incubated with the cells for a period of time to allow them to break open all the cells. With some systems (like plasmid preparation from bacteria) this can be very fast at maybe 5 minutes, whilst in others (like digesting tissues to extract genomic DNA) it can take an overnight incubation and a temperature the enzyme likes (e.g. 55ºC). 3. Once the contents of the cells are liberated, the DNA you want must be separated from all the bits you don't want. The easiest way to do this is to precipitate the DNA out from the rest using a salt (sodium from sodium acetate is one of the most common) and alcohol. How the salt and alcohol do this I have been unable to find in any of my textbooks (it's just something we've always done it seems!), but I'm sure any halfway competent chemist probably knows. Two answers I did find that might have some merit were: - "The positively charged sodium, ammonium, lithium or potassium ions used in ethanol precipitations shield the negative charge on the phosphate backbone of the DNA or RNA strand. As a result, non-ionic, hydrophobic interactions take place and provoke aggregation of the nucleic acids in ethanol or isopropanol." (This statement was by Tim Fitzwater) - an idea supplied by Paddy was that the salt ions formed a bond with the DNA and that the salt-DNA molecule was insoluble in alcohol. 4. Finally the DNA is then centrifuged down and washed in 70% ethanol to remove the residual salt. Hey presto - DNA! Hope that helps ya. Adrian
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