COLD SPRING HARBOR, N.Y. (Wednesday, April 1, 2009) – Along with new cutting-edge methods, Cold Spring Harbor Protocols is home to an in-depth library of basic laboratory methods. The April issue (www.cshprotocols.org/TOCs/toc4_09.dtl) features two of these standard techniques.
From molecular biology researchers to law enforcement forensics laboratories, polymerase chain reaction (PCR) is the commonly used method for nucleic acid amplification. But PCR is often difficult to optimize, and failure to do so can lead to undefined and unwanted products, or a complete lack of amplification altogether. To help avoid these issues, Kenneth Roux from Florida State University (http://bio.fsu.edu/~roux/) provides "Optimization and Troubleshooting in PCR." The article addresses various optimization strategies including touchdown PCR and hot-start PCR. Magnesium concentration, buffer pH, and cycling conditions are also considered. The article is freely accessible on the website for "Cold Spring Harbor Protocols" (http://cshprotocols.cshlp.org/cgi/content/full/2009/4/pdb.ip66).
The loading of individual cells with fluorescent probes via patch pipettes is regularly performed in neuroscience research laboratories. This method allows for combined electrophysiological and optical measurements at a quantitative level. Arthur Konnerth and colleagues from the Institut für Physiologie der Ludwig-Maximilians-Universität München (http://www.imprs-ls.de/professors/konnerth.htm) have written "Dye Loading with Patch Pipettes," available in the April issue. This patch-clamp methodology has been successful for single-cell dye labeling in cultured neurons, brain slices, and in vivo preparations. A wide range of dyes can be loaded using this method, including probes for morphological reconstruction, ion-sensitive indicator dyes for monitoring second-messenger cascades, and dye-labeled proteins for fluorescence resonance energy transfer (FRET), fluorescence correlation spectroscopy (FCS), and fluorescence recovery after photobleaching (FRAP) studies. The most widespread application of this technique has been for Ca2+ imaging. This method is freely accessible on the website for Cold Spring Harbor Protocols (http://cshprotocols.cshlp.org/cgi/content/full/2009/4/pdb.prot5201).
