MadSci Network: Molecular Biology
Query:

Re: Why are the ingredients in a DNA extraction important?

Date: Tue May 23 09:24:20 2000
Posted By: Adrian Hardy, Staff, Development, Imutran Ltd
Area of science: Molecular Biology
ID: 955319984.Mb
Message:

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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