isolation of e. coli chromosomal dna lab report

isolation of e. coli chromosomal dna lab report - Isolation...

Info iconThis preview shows pages 1–4. Sign up to view the full content.

View Full Document Right Arrow Icon
Isolation of E. Coli Chromosomal DNA
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Introduction E. coli is known as "enteric bacteria", because these bacteria primarily inhabit the intestinal tract of humans as well as certain animals. E. coli belongs to the bacterial family Enterobacteriaceae, which consists of nonsporeforming, rod-shaped, Gram-negative bacteria which move by using flagella. Regardless of presence or absence of oxygen, most strains develop very well on the usual laboratory media. E.coli may often contain small circular DNA molecules called plasmids of 1,000‐200,000 DNA base pairs. Although the plasmids are extra chromosomal, in other words not part of the bacterial genome, they do contain genetic information. Replication may occur either when bacterial cells replicates or independently from cell division. Genetic information available to the bacterial cell can be altered and transformed through the use of plasmids. In cases when multiple genes are contained in plasmids, the genes are always transferred collectively. E. coli possess the ability to grow both aerobically and anaerobically in the intestinal environment they dwell in. E. coli are well accustomed to such environments by the fact that they live off a relatively limited number of low-molecular weight substances, available in relatively low concentrations and, usually, momentarily. In the laboratory, much as in their natural habitat, E. coli react quickly to factors as chemicals, pH, temperature, or osmolarity. They move towards or away from changes in levels and presence of chemicals and gases in their environment. E. coli is extremely efficient in the production of enzymes for degradation of carbon sources as well enzymes for synthesis of metabolites. Such enzymes are generated only if sufficient sources are available in the natural environment.
Background image of page 2
In the laboratory experiment, the foremost step is to cause cell lysis after cells have been grown and concentrated in a microcentrifuge tube. The cells are be resuspended through the use of a buffer holding Tris and EDTA. Divalent cations as Ca 2+ and Mg 2+ are involved in stabilizing the membrane of E. coli and the EDTA helps to bind them, and, thus, destabilize the bacterial membrane. Afterwards, we add a solution of NaOH and the detergent SDS (sodium dodecyl sulfate). The latter assists in making the membranes more soluble and causes the bacterial cells to lyse. This is an observable and easily identifiable physical change, involving protein precipitation and viscosity increase. This, in brief, is step one. The second step involves gel electrophoresis, which is a technique of separating molecules through a porous semisolid matrix based on their different rates of agility in an applied electric field. The semisolid matrix used in the experiment is essentially a gel from agarose and buffered water. Movement of molecules through the agarose gel is dependent on their length and charge and the sizes of the pore gel itself. When an electric field is applied across the gel, proteins tend to migrate to the positive electrode (anode) at neutral pH, DNA,
Background image of page 3

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Image of page 4
This is the end of the preview. Sign up to access the rest of the document.

Page1 / 9

isolation of e. coli chromosomal dna lab report - Isolation...

This preview shows document pages 1 - 4. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online