Tissues and Tissue Culture Cells

In contrast to tissues, tissue culture cells are readily lysed with detergent [11]. Adherent cells from a culture plate are first scraped using a rubber policeman into a small volume of phosphate-buffered saline (137 mMNaCl, 2.7 mM KC1, 4.3 mM Na2HP04, 1.4 mM KH2P04, pH 7.3) and harvested by centrifugation at 1500 x g for 10 minutes at 4°C. The cellular pellet is resuspended in ice-cold TE so that 1 mL contains 100 million cells. After the addition of 10 volumes of freshly prepared digestion buffer (10 mM Tris-Cl, pH 8, 0.05 EDTA, pH 8, 0.5% Sarcosyl, and 100 pg/mL proteinase K), the sample is incubated at 50°C for 3 hours. DNA is recovered by ethanol precipitation after extraction with phenol, phenol-chloroform, and chloroform as described earlier. 

Polymerase chain reaction (PCR) is a method for amplifying DNA from a small amount of DNA catalyzed by thermostable DNA polymerase under appropriate reaction conditions with a pair of primers (oligonucleotides) that are complementary to DNA. K. Mullis, who invented the technique in the 1980s, was awarded a Nobel prize in 1994. Since its invention, various refinements and modifications have been described, and several review articles and books have been written on the subject [17-20]. 

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Sackett, D. L. (1995). Rapid purification of tubulin from tissue and tissue culture cells using solid-phase ion exchange. Anal. Biochem. 228, 343-348. 

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