DNA Amplification

PCR & Reaction Cleanup

Purification of DNA from a PCR reaction is typically necessary for downstream use, and facilitates the removal of enzymes, nucleotides, primers and buffer components. Traditionally this was accomplished using organic extraction methods, such as phenol chloroform extraction, followed by ethanol precipitation. With this method, the PCR reaction is mixed with a phenol-chloroform mixture. The organic phase then separates from the aqueous phase, taking with it any proteins or enzymes that were in the original sample. The amplified DNA remains in the aqueous layer of the mixture, along with other reaction components (e.g., buffers, nucleotides and primers). The desired DNA in the aqueous phase can then be separated from other contaminants by ethanol precipitation.

Today, there are faster and less hazardous methods available. Commonly used methods employ spin columns containing a silica matrix to which DNA can be selectively bound in the presence of chaotropic salts. Other reaction components, such as enzymes, nucleotides, detergents, and primers either do not bind well or are removed during the wash steps. Finally, the DNA is eluted from the columns under low-salt conditions and is ready for demanding downstream applications.

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