E-Book Overview
An essential core collection of the latest molecular and genetic techniques for cloning, subcloning, sequencing, PCR, protein expression, and much more. Each protocol represents a time-tested, step-by-step recipe that creates an understanding of the procedure, easily reproducible results, and confidence that the procedure will work. The collection includes not only many updated and improved classic techniques, but also a powerful group of advanced methods that point to future progress, among them nonisotopic DNA labeling, silver staining, and automatic sequencing. This excellent bench companion will help those who need to learn for the first time how to conduct research on the molecular biology of nucleic acids or those who need to broaden their competence and laboratory skills. Even highly skilled researchers will find many time-saving techniques.
E-Book Content
CHAPTER1
The Simultaneous Isolation of RNA and DNA from Tissues and Cultured Cells Frank Merante, Sandeep Juta K. Reed, and Gerald
Raha, Proteau
1. Introduction Many techniques are currently available that allow the isolation of DNA (I-7) or RNA (8-231, but such methods allow only the purification of one type of nucleic acid at the expense of the other. Frequently, when cellular material is limiting, it is desirable to isolate both RNA and DNA from the same source. Such is the case for biopsy specimens, primary cell lines, or manipulated embryonic stem cells. Although several procedures have been published that address the need to simultaneously purify both RNA and DNA from the same source (2&31), most methods are simply a modification of the original procedure of Chirgwin et al. (8). Such procedures utilize strong chaotropic agents, such as guanidinium thiocyanate and cesium trifluoroacetate (25,2 7), to simultaneously disrupt cellular membranes and inactivate potent intracellular RNases