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Extraction of DNA from Agarose gel
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Objective

 

To extract specific bands of  DNA from agarose gels in which they are  separated through electrophoresis.


 

Theory


DNA isolation is a critical step in molecular biology. It is necessary to obtain a specific DNA fragment from the extracted DNA in molecular biology techniques. After isolating plasmids it may contain some chromosomal DNA contamination it will interrupt the further processing of cloning. So it is better to recover the plasmid DNA by eluting it from agarose gels (extraction). The first step in extracting DNA is identifying the DNA band which is to extract, by illuminating under UV light. The desired band is then carefully cut by a Scalpel blade. There are several methods for extracting DNA from the agarose gels. Recovery of DNA from agarose gels by electrophoresis onto DEAE-cellulose membrane is one of the rapid and effective methods. Electroelution is also a good method for DNA recovery especially for larger DNA fragments. Several kit methods are also used in laboratories. In electro elution, the gel fragment of desired DNA band is placed into a dialysis bag with buffer. The bag is then placed into a gel box containing buffer and subjected to an electric current. The DNA extracted is precipitated from the solution. In another recovery method using DEAE cellulose membrane, the gel piece is slide into the slit of DEAE cellulose paper which will bind the DNA. Then an electric current is applied in order to move the band in the paper. DNA is washed off from the paper and is precipitated with ethanol. Freeze-thaw method of extraction is a commonly used advantageous DNA recovering method which will supports the common laboratory facilities. It is very simple and easy to perform with good yield.


Most of the molecular biology laboratories use Low melting point agarose for the separation of DNA from agarose. Low melting point agarose melts at a lower temperature than standard agarose and this temperature does not denature double stranded DNA. It is better to extract DNA fragments in a TAE buffered gel than TBE buffered gel because borate present in the buffer interferes with purification methods.

 

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