Question

After the blue/white screen, you will isolate plasmid from chosen colonies, and confirm the presence of the PCR product by restriction enzyme digestion. There is an EcoRI restriction site flanking the multiple cloning site of the plasmid. Therefore, by digesting the purified plasmid with a single restriction enzyme, you can remove the PCR product from the plasmid. If the plasmid truly contains a PCR product, then you will be able to visualize the PCR product and plasmid as separate bands on an agarose gel. .

Answer 1

Polymerase chain reaction (PCR) is a method widely used in molecular biology to make many copies of a specific DNA segment. Using PCR, a single copy (or more) of a DNA sequence is exponentially amplified to generate thousands to millions of more copies of that particular DNA segment. The most widely used method for analyzing the PCR product is the use of agarose gel electrophoresis, which separates DNA products on the basis of size and charge. Agarose gel electrophoresis is the easiest method of visualizing and analyzing the PCR product.

Thus, an uncut plasmid produces two bands on a gel, representing the oc and ccc conformations. If the plasmidis cut once with a restriction enzyme, however, the supercoiled and open-circular conformations are all reduced to a linear conformation.

slots 2

Although 16S rRNA gene sequencing is highly useful in regards to bacterial classification, it has low phylogenetic power at the species level and poor discriminatory power for some genera and DNA relatedness studies are necessary to provide absolute resolution to these taxonomic problems. The genus Bacillus is a good example of this. The type strains of B. globisporus and B. psychrophilus share >99.5% sequence similarity with regard to their 16S rRNA genes, and yet at the DNA level exhibit only 23 to 50% relatedness in reciprocal hybridization reactions. In our laboratory we have found that the type strains of Edwardsiella species exhibit 99.35 to 99.81% similarity to each other, and yet these three species are clearly distinguishable biochemically and by DNA homology (28 to 50% relatedness). Such examples indicate that SSU sequence similarity even to a very high level does not in each case imply identity or accuracy in microbial identifications. Many investigators have found resolution problems at the genus and/or species level with 16S rRNA gene sequencing data. These groups include (not exclusively), the family Enterobacteriaceae (in particular, Enterobacter and Pantoea), rapid-growing mycobacteria, the Acinetobacter baumannii-A. calcoaceticus complex, Achromobacter, Stenotrophomonas, and Actinomyces. Some of these problems are related to bacterial nomenclature and taxonomy while others are related to different issues cited. A further problem regarding the resolution of 16S rRNA gene sequencing concerns sequence identity or very high similarity scores. Reports have documented 16S rRNA gene sequence similarities or identity for the Streptococcus mitis group and other nonfermenters. In such instances 16S rRNA gene sequence data cannot provide a definitive answer since it cannot distinguish between recently diverged species. In other instances, the difference between the closest and next closest match to the unknown strain is <0.5% divergence (>99.5% similarity). In these circumstances, such small differences cannot justify choosing the closest match as a definitive identification, although in some studies this is exactly what was done .