Biotech 2nd Edition Sample Ch. 4 Biotechnology Sample Ch. 4 - Page 20

Laboratory 4h DNA Extraction from Bacteria Background Within bacteria cells, DNA and, therefore, genes are found both in the single strand of genomic DNA and in any extrachromosomal plasmids. Either source of DNA may be a source of genes for genetic engineering or gene therapy purposes. Isolation of either type of DNA includes bursting open the cells, getting rid of contaminant molecules, and precipitating the DNA out of the solution. To burst open cells, the cell membrane must be removed. Since the major component of a membrane is a phospholipid bilayer, a detergent can be used to dissolve away this “fatty” layer. Several different detergents may be used depending on the type of DNA to be extracted. For bacteria cells, SDS works well. Even household detergents, such as Dawn® and Ivory® (both manufactured by Procter & Gamble), have been used. When the cells burst, all the cellular contents are released into the collection vessel. The detergent not only removes the lipids, but also precipitates many of the proteins from the membrane and cytoplasm. These proteins drop to the bottom of the vessel and are easy to separate from the DNA. Adding heat speeds the process. Enzymes may be added to degrade other molecules. For example, RNase is commonly used to decompose RNA contaminant molecules in DNA extractions. Proteases are used to degrade protein contaminants in samples. Centrifugation can separate the precipitated proteins and degraded cellular debris from the DNA still in solution. The DNA can be drawn out of solution by adding alcohol, usually ethanol (EtOH), or isopropanol. If genomic DNA is desired, the DNA can be spooled onto a glass rod (see Figure 4.14) or it can be pelleted by a high-speed centrifuge. Plasmid DNA is much too small to be spooled. It is precipitated from solution using a series of alcohol washes and centrifugation. Ultimately, plasmid DNA is recovered from one of the pellets left after one of the centrifugations. Plasmid isolation is presented in Chapter 8. Purpose Can relatively pure genomic DNA be extracted from E. coli bacteria cells? Hold the container and rod so the layers are parallel to the floor. EtOH bubbles trapped in DNA strands aqueous DNA layer Figure 4.14.  Spooling Technique.  When spooling, hold the glass rod almost parallel Twirl and scoopthe at the between the DNA threads out and to the floor. Rotate and revolve through twointerface layers, scooping the alcohol and aqueous (watery) DNA layer. spinning them around the rod. DNA Isolation and Analysis 85