Advanced Concepts

Sometimes, DNA preparations are intended for long-term storage. The presence of the chelating agent EDTA protects the DNA from damage by DNAses present in the environment. EDTA is a component of TE buffer (10 mM Tris, 1 mM EDTA) and other resuspension buffers. The EDTA will also inhibit enzyme activity when the DNA is used in various procedures such as restriction enzyme digestion or polymerase chain reaction (PCR). One must be careful not to dilute the DNA too far so that large volumes (e.g., more than 10% of a reaction volume) of the DNA-EDTA solution are required. When DNA yield is low, as is the case with some clinical samples, it is better to dissolve it in water. More of this can be used in subsequent procedures without adding excess amounts of EDTA. Because the entire sample will be used for analysis, protection on storage is not a concern.

therefore, been developed and are used in many laboratories. Initially, these methods did not provide the efficient recovery of clean DNA achieved with phenol extraction; however, newer methods have proven to produce high-quality DNA preparations in good yields.

Inorganic DNA extraction is sometimes called "salting out" (Fig. 4-2). It makes use of low pH and high salt conditions to selectively precipitate proteins, leaving the DNA in solution. The DNA can then be precipitated as described above using isopropanol pelleted and resus-pended in TE buffer or water.

Was this article helpful?

0 0

Post a comment