Combining Techniques to Answer Molecular Questions

This manual is a collection of basic techniques central to the study of nucleic acids, proteins, and whole-cell/subcellular structures. The following is an overview of how the basic techniques described in this manual can be used alone or in sequence to answer questions about the properties of proteins and nucleic acids. Flowcharts are provided to orient the novice researcher in the use of fundamental molecular techniques, and provide perspective regarding applications of the technical units in this manual.

Listed below are common questions about nucleic acids and techniques used to answer them. Also refer to Figure 1.

Listed below are common questions about proteins and techniques used to answer them. Also refer to Figure 2.

For many experiments, the concentration of protein in the sample must first be quantified ( UNIT here). For example, this is often done prior to performing SDS-PAGE and/or an immunoblot to ensure equal loading of different protein samples for comparison. To determine the localization of specific proteins, cells can first be lysed and fractionated by centrifugation ( UNIT here), followed by immunoblotting of the proteins ( UNIT here) from fractions containing specific cell substructures. A chromatography step would further resolve proteins from the various fractions ( UNITS here & here). Alternatively, localization of specific proteins to distinct cellular structures can be done using the immunofluorescence technique ( UNIT here).

This manual also includes techniques for studying whole cells and their substructures. These include cell fractionation by centrifugation ( UNIT here), cell imaging by conventional light microscopy ( UNIT here), and imaging by fluorescence microscopy ( UNIT here). Refer to Figure 3. All of these protocols require first growing cells in culture. Protocols for culturing bacteria ( UNIT here), culturing mammalian cells ( UNIT here), and culturing yeast ( UNIT here) are provided.

These techniques can be used to answer questions such as:

Does cell morphology change under different treatment conditions?

Does cell behavior change under different treatment conditions?

Do genetically altered cell lines display morphological phenotypes?

In which cellular substructures does a specific endogenous or altered protein reside?

Conventional light microscopy can be used to image most cell organelles and structures by using the appropriate microscopy technique. Common variations and their applications are described in UNIT here. Fluorescence microscopy is used to image specific organelles with fluorescent dyes, or to study the localization of specific proteins ( UNIT here).

For any experiment performed, it is essential to keep thorough records in the form of a laboratory notebook. APPENDIX here outlines the best practices for organizing and recording experimental details to optimize their usefulness and completeness.

Results from many techniques in this manual require digital imaging for documentation in a laboratory notebook and for publication. APPENDIX here and APPENDIX here present important ethical and practical considerations for capturing, manipulating, and storing digital images, as well as guidelines for preparing them for publication.

Some experimental results will require statistical analyses. APPENDIX here provides guidelines for selecting and using appropriate statistical tests in the life sciences.