One of the many advantages to using yeast as a model system is that large quantities of biomacromolecules, including nucleic acids (DNA and RNA), can be purified from the cultured cells.
This video will address the steps required to carry out nucleic acid extraction. We will begin by briefly outlining the growth and harvest, and lysis of yeast cells, which are the initial steps common to the isolation of all biomacromolecules. Next, we will discuss two unique purification methods for the separation of nucleic acids: column binding and phase separation. Additionally, we will demonstrate several ways in which these methods are applied in the laboratory, including the preparation of nucleic acids for molecular biology techniques such as PCR and southern blotting, quantification of gene expression in response to environmental stimuli, and purification of large amounts of recombinant proteins.…
Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - Arizona University
Demonstrating Author: Luisa Ikner
The spectrum of research in environmental microbiology is broad in scope and application potential. Whether the work is bench-scale with known bacterial isolates, or in the field collecting soil or water samples containing unknown bacterial isolates, the ability to quickly and visually discern culturable populations of interest remains of great import to environmental microbiologists even today with the abundance of molecular techniques available for use. This video will demonstrate one such technique, known as Gram staining.…
Transformation is the process that occurs when a cell ingests foreign DNA from its surroundings. Transformation can occur in nature in certain types of bacteria. In molecular biology, transformation is artificially reproduced in the lab via the creation of pores in bacterial cell membranes. Bacterial cells that are able to take up DNA from the environment are called competent cells. In the laboratory, bacterial cells can be made competent and DNA subsequently introduced by a procedure called the heat shock method.
Heat shock transformation uses a calcium rich environment provided by calcium chloride to counteract the electrostatic repulsion between the plasmid DNA and bacterial cellular membrane. A sudden increase in temperature creates pores in the plasma membrane of the bacteria and allows for plasmid DNA to enter the bacterial cell. This video goes through a step-by-step procedure on how to create chemically competent bacteria, perform heat shock transformation, plate the transformed bacteria, and calculate transformation efficiency.…
Basic Methods in Cellular and Molecular Biology
S. cerevisiae are unicellular eukaryotes that are a commonly-used model organism in biological research. In the course of their work, yeast researchers rely upon the fundamental technique of transformation (the uptake of foreign DNA by the cell) to control gene expression, induce genetic deletions, express recombinant proteins, and label subcellular structures.
This video provides an overview of how and why yeast transformation is carried out in the lab. The important features of yeast plasmids will be presented, along with the procedure required to prepare yeast cells to incorporate new plasmids. The presentation also includes a step-by-step protocol for the lithium acetate method of yeast transformation. Finally, examples of the many applications of this essential technique will be provided. …
Source: Roberto Leon, Department of Civil and Environmental Engineering, Virginia Tech, Blacksburg, VA
Wood is a ubiquitous material that has been used in construction from the earliest times. Wood is a renewable, sustainable material with great aesthetic value. Today, there are probably more buildings constructed with wood than any other structural material. Many of these buildings are singlefamily residences, but many larger apartment buildings, as well as commercial and industrial buildings, also use wood framing.
The widespread use of wood in construction has appeal from both an economic and aesthetic basis. The ability to construct wood buildings with a minimal amount of equipment has kept the cost of woodframe buildings competitive with other types of construction. On the other hand, where architectural considerations are important, the beauty and warmth of exposed wood is difficult to match with other materials.
The objectives of this experiment are to conduct tensile and compressive tests on three types of wood to investigate their stress-strain behavior, and to conduct a four-point bending test on a wood beam to ascertain its flexural performance. In a four-point bending test, a simply-supported beam is loaded with two equal point loads at its third point…
Plasmid purification is a technique used to isolate and purify plasmid DNA from genomic DNA, proteins, ribosomes, and the bacterial cell wall. A plasmid is a small, circular, double-stranded DNA that is used as a carrier of specific DNA molecules. When introduced into a host organism via transformation, a plasmid will be replicated, creating numerous copies of the DNA fragment under study.
In this video, a step-by-step generalized procedure is described for how to perform plasmid purification. Plasmid purification includes three basic steps: growth of the bacterial culture, harvesting and lysis of the bacteria, and purification of the plasmid DNA. The video contains an explanation where the plasmid can be found in each step of the protocol and to quantitatively and qualitatively analyze plasmid DNA with a spectrophotometer and/or gel electrophoresis. There are different types of plasmid purification methods available, which are geared toward desired yield, plasmid copy number, and bacterial culture volume.…
Basic Methods in Cellular and Molecular Biology