PlasmidsBacterial HostsMeasuring FluorescenceExperiment 8 – Transformation of E.coliThe Components of the PCR ReactionProtocolDNAMaking Buffers in a Biochemistry LabMaking the Buffer - TheoryThe Concentration of the BufferReagentsAgarose GelsAmpicillin Solution (1000X)BuffersBradford Reagent for Protein AssayDNA Denaturing SolutionDNA Loading Solution (10X)DNA Neutralization SolutionHoescht Assay SolutionIPTG Solution (100X)PNPG SolutionsSDS Loading Buffer (10X)SDS Gel Electrode Buffer (10X)Separating Gel SolutionSSC (20X)Stacking GelSubstrate Solution for BlotsTAE Buffer (10X)TE BufferTetracyclin Solution (1000X)TNE Solution – (2X)Towbin BufferTSS SolutioneXgal Stock SolutionXgal Agar PlatesYT MediumZ BufferINTRODUCTION TO NUCLEIC ACIDS AND MOLECULAR CLONINGReferences: - E.coli: Cellular and Molecular Biology: F.C. Neidhardt ed.Volumes 1,2. (1987)- a detailed reference on things E.colial.- www.slic2.wsu.edu:82/hurlbert/micro101/pages/101hmpg.html an excellent review of most of the things we will do in the lastsection of the lab. A MUST-READ-BEFORE-DOING-AN-EXPERIMENT website.- Voet, Biochemistry, pp916-918, 932-933, 897-902.- Jacobs, “Life in the Balance- Cell Walls…” Science, 278, 1731(1997)The next series of experiments will be devoted to the nucleic acids. In them se we are going to learn some of the basic techniques that are usedby biochemists who study nucleic acids. At one time, nucleic acid research was a separate branch of the field of biochemistry practiced mainly by microbiologists and a few hardy organic chemists and physicists. Researchers could and did study enzymes without knowing much about how to work with nucleic acids, except perhaps how to get rid of them in the early steps of enzyme purification. This is no longer so.The advent of recombinant DNA technology, a technology which includes the discovery of restriction endonucleases i.e., the ability to cut at specific DNA sites, thus enabling us to cut out defined chunks of DNA, thediscovery of methods of inserting foreign DNA into other organisms and also of having them expressed in quantity, has given biochemists a useful and general way of producing large quantities of almost any DNA, RNA and protein they want to study. It has also provided a way to produce modified enzymes on demand. This makes it possible to test models for catalysis, for example, by making an enzyme that lacks a particular amino acid in the active site.The experiments in the DNA section of the course cover some of the basic methods used in recombinant DNA work. Your instructors have not been able to reduce the content of some of recombinant DNA protocols so that they can be completed in a single afternoon. Therefore,you may sometimes be required to come in early to get an experiment started. Labs sometimes stretch over several lab periods so you may be required to come in on an off day to get things ready for the next session.We have, however, been able to adapt a good cross-section of useful techniques to a suitable time frame and those of you who may be going on to do further work in biochemistry should find this a useful exercise.Plasmids1Chromosomal DNA contains the biological information we need. Chromosomal DNA is difficult to obtain in pure form and, if allowed unrestricted transcription and translation would produce a large mass of biochemical products. Thus, if we want to use chromosomal DNA as a source from which we produce our material of interest, we must painfully remove the very small fraction of the components of interest from a larger mass of irrelevant products with very similar chemical and physicalproperties. Historically, the solution to this problem has been to study smaller pieces of DNA as a substitute for chromosomal DNA. Ideally, small pieces need to be constructed so that they not only contain the component we want to examine, but also DNA components that will allowthem to be replicated by cellular machinery. An example of such a piece of DNA is called a plasmid. Plasmids are extrachromosomal, semi-autonomously-replicating pieces of DNA that occur naturally in bacterial cells. They allow genetic information to be transferred both vertically and horizontally. Plasmids are circular, and contain genetic elements thatallow replication in a suitable host by using the enzymes and energy provided by the host. In short, plasmids are molecular parasites. Like viruses they use cells for propagation of the species. They usually provide a benefit for the host by carrying genes that might confer selective advantages to the host. Some of these selective advantages are providing genes for antibiotics resistance, for trace element metabolism, or that enhances disease-causing ability.One of the most widely used plasmids in the laboratory is called pBR322. It is a circular bit of DNA of about 4600 base pairs. The designation "pBR" derived from the first letters of "plasmid Bolivar Rodriguez" the latter two letters being the initials of the constructors of the plasmid-Bolivar and Rodriguez. pBR322 is derived from a much larger natural plasmid, and has been stripped down to the bare minimumof sequence required to carry out its laboratory functions. This plasmid has two protein-coding genes, one that confers resistance to ampicillin, aform of penicillin, while the other confers resistance to tetracycline. The presence of these genes allow bacteria which carry them, to grow on nutrients containing those antibiotics, whereas ordinary bacteria lacking that gene will not grow. This process of growing bacteria under conditions where only a desired genotype is viable is called “selection”. We will make extensive use of selection to isolate recombinant species ofinterest. There is also an origin of replication, which governs plasmid replication. Plasmids such as pBR322 are replicated by a "relaxed" or “low stringency” mode. These plasmids can replicate without host protein synthesis requiring only DNA polymerase I activity. “Relaxed replication” plasmids are present in about 10-100 copies per cell and are passed to each daughter cell as the parent cell divides.Unlike most natural plasmids, pBR322 and its derivatives have no system for spreading horizontally through the bacterial population; they 2cannot be transferred by bacterial conjugation. The use of non-conjugative plasmids is a sort of a safety feature, so those antibiotic-resistant traits cannot easily and inadvertently, be transferred to