1. (a)
I. Plasmids are important tools in molecular biology. Plasmids are small circular DNA that has the ability to enter and replicate in bacterial cells and can be used as vectors to introduce foreign genes into bacteria for cloning and sequencing. Any gene must be inserted into an appropriate location of a plasmid to be expressed. The importance of a plasmid is in the step of cloning and sequencing when the construction of a recombinant DNA molecule occurs. The target gene fragment is ligated to a plasmid, and becomes recombinant DNA. Then the plasmid can replicate autonomously in an appropriate host organism.
II. The polymerase chain reaction (PCR) is the amplification of DNA sequence by repeated cycles of strand seperation and replication. This is a direct method of making copies of a desired DNA sequence, unlike the technique using plasmids. PCR is a process quite like DNA replication. It is still the process of two DNA strands unwinding, replicating, and then reannealing, however the strands are separated by heat. Generally temperatures must be increased to 94-96 degrees C for the hydrogen bonds to break and the separation to occur. Once the stands are separated they can be used as templates for complementary strands to be synthesized by DNA primers. After the strands are completely synthesized, the temperatures are brought back down to 50-65 degrees C for the primers to anneal with the template DNA, and a DNA polymerase can build complementary strands using free nucleotides that have been added to the solution.
III. Restriction fragment length polymorphism (RFLP) analysis is a technique in which DNA regions are digested using restriction endonucleases, and subjected to radioactive complementary DNA probes to compare the differences in DNA fragment lengths between individuals. The DNA in question is digested using restriction endonuclease(s). The DNA is then run on a gel and appears to be very long. The gel is subject to a chemical that causes the double-stranded DNA to separate into to individual strands. The strands are then transferred to a nylon membrane with using an electric current, where it will bind. The transfer process is called Southern blotting.
Once the recombinant plasmid was obtained, it was then inserted into E. coli cells through transformation. From a successful transformation, we expected the bacterial cells to translate the inserted EGFP sequence into its protein form. The bacteria cultures were plated on petri dishes containing growth supplement, Luria Broth (LB), an antibiotic: Kanamycin, and IPTG which induced the fluorescence property within successfully transformed bacterial colonies. Different variants of the petri dishes were also included as control and unknown.
The plasmids in lanes 3,4,8 and 9 have been digested using one restriction enzyme and had been cut at one restriction site, resulting in a linear molecule. Comparing lanes 3 and 4 to
Recombinant DNA technology: Sub cloning of cDNA molecule CIH-1 into plasmid vector pUC19, transformation of XLI-Blue Ecoli & restriction mapping.
al. (1994) explain that a complementary DNA for GFP produces a fluorescent product when expressed in E. coli cells as the expression of GFP can be used to monitor gene expression and protein localization in living things. In this experiment, the heat shock method will be used to deliver a vector (plasmid) of GFP to transform and grow E. coli bacteria. Four plates containing Luria Bertani (LB) broth and either –pGLO or +pGLO will have E. coli bacteria added to it. The plate containing –pGLO (no pGLO) and LB will show growth as ampicillin will be present killing bacteria but no glowing because no arabinose will be present for glowing to be activated, the same result will be seen in the plate containing +pGLO, LB and ampicillin.
Amplification reaction was done in a 25.0 µL reaction mixture containing 0.4 µL DNA (from DNA extraction), 5.0 µL of 10X PCR reaction buffer, 14.2 µL of sterelized dH2O, 2.0 µL of magnesium chloride (MgCl2, 25 mM), 1.0 µL nucleotide/dNTP mix (10 Mm), and 0.4 µL of 5 u/µL Taq DNA polymerase for each primer namely respectively. The components and the volume used for the amplification reactions are listed in Table 3.2. For the reaction, PCR reaction was performed in a programmable gradient-enabled thermocycler (Bio-Rad MyCycler™ Thermal Cycler).
PCR or polymerase chain reaction is not a DNA typing technique, but a variety of different DNA tests (Riley). PCR duplicates and increases the quantity of a DNA strand which is beneficial to forensic scientists who are faced with little quantity of materials (Saferstein 394). The introduction of PCR-based testing in DNA analysis required scientists to switch to smaller targets that had the same repetitive variation (Jones). This is how short tandem repeat, the newest method of DNA typing,
The two modes of analysis that will be used to identify an unknown insert piece of DNA would be plating the transformation cells onto LA plates that have either ampicillin or chloramphenicol and PCR. We will use the PCR thermocycler to denature the restriction enzymes that were specifically used to assimilate the vector DNA. It is important to use the PCR thermocycler because denaturation of the restriction enzyme will prevent the restriction enzyme from cutting the vector DNA, after the insert DNA has assimilated to the vector DNA. After the addition of specific primers that complement the base pair to its corresponding target strand, PCR will be used. Subsequently, Taq polymerase will be used to determine whether the insert DNA has been properly assimilated to the vector DNA. Within this specific situation, the target strand will be the insert DNA. After we let the PCR thermocycler run for approximately 2 ½ hours, we will then put our PCR products in the gel and run the gel to completion. After the gel has run to completion, we will then take a photograph of the gel using the UV transilluminator with the assistance of our TA. If the insert DNA was properly assimilated to the vector DNA, then our corresponding gel photo would have one band. After the cells have been transformed, we would g...
The synthetic A and B chains are then inserted into the bacteria’s gene for B-galactosidase, which is carried in the vectors plasmid. The vector for the production of insulin is a weakened strain of the common bacteria Escherichia coli, usually called E. coli. The recombinant plasmids are then reintroduced to the E. coli cells. As the B-galactosidase replicates in a cell undergoing mitosis the insulin gene is expressed. To yield substantial amounts of insulin millions of the bacteria possessing the recombinant plasmid are required.
Imagine if your pet was getting experimented on for a product you might buy in the future. Would anyone really want that product, your pet was in pain because of it? Animals are getting experimented on for products to get released to the public. Some companies are using vitro researching to test their products but not enough companies are using vitro as their form of testing products. Synthetic skin could reduce the amount of animals getting tested on everyday for companies to release new products to the public. Animals are getting experimented on everyday.
Over the last fifty years or so, scientists have made a great amount of progress in this area, including the development of techniques which allow for the controlled manipulation and replication of specific segments of the human genome. These types of techniques have come to be known as recombinant DNA (rDNA) technology and have allowed scientists to analyze functions of genes which are not necessarily directly expressed at the phenotypic level. This is done by "cutting out" or excising a particular segment of DNA of interest from the genetic material of an individual and inserting it into a bacterial plasmid (a tiny ring of DNA in addition to the normal chromosomal material found within the cells of bacteria).
Biology literally means "the study of life". Biology is such a broad field, covering the minute workings of chemical machines inside our cells, to broad scale concepts of ecosystems and global climate change. Biologists study intimate details of the human brain, the composition of our genes, and even the functioning of our reproductive system. Biologists recently all but completed the deciphering of the human genome, the sequence of deoxyribonucleic acid (DNA) bases that may determine much of our innate capabilities and predispositions to certain forms of behavior and illnesses. DNA sequences have played major roles in criminal cases (O.J. Simpson, as well as the reversal of death penalties for many wrongfully convicted individuals), as well as the impeachment of President Clinton (the stain at least did not lie). We are bombarded with headlines about possible health risks from favorite foods (Chinese, Mexican, hamburgers, etc.) as well as the potential benefits of eating other foods such as cooked tomatoes. Informercials tout the benefits of metabolism-adjusting drugs for weight loss. Many Americans are turning to herbal remedies to ease arthritis pain, improve memory, as well as improve our moods. Can a biology book give you the answers to these questions? No, but it will enable you learn how to sift through the biases of investigators, the press, and others in a quest to critically evaluate the question. To be honest, five years after you are through with this class it is doubtful you would remember all the details of meatbolism. However, you will know where to look and maybe a little about the process of science that will allow you to make an informed decision. Will you be a scientist? Yes, in a way. You may not be formally trained as a science major, but you can think critically, solve problems, and have some idea about what science can and cannoit do. I hope you will be able to tell the shoe from the shinola.
For centuries scientists have used animals to study the causes of diseases; to test drugs, vaccines and surgical techniques; and to evaluate the safety of chemicals used in pesticides, cosmetics and other products. However, many scientists amongst animal- right activists forbid the use of animals in scientific research regardless how many illnesses are eliminated through the use of animals in scientific research. Amongst animal right activists, David Suzuki also raises concerns towards animal experimentation. In his article, The Pain of Animals, Suzuki argues that humans have no right to exploit animals because--much like humans--animals also experience pain. In contrast to Suzuki, Haldane, in his article, Some Enemies of Science, argues because animals are very similar to humans, scientists have no choice but to use animals in scientific experiments. Both authors greatly contrast their opinions towards animal experimentation; however Haldane has a more explanatory approach towards animal experimentation. He argues animal experimentation should be acceptable because other forms of animal exploitation are acceptable in society. Secondly, unlike other forms of exploitation which seek pleasure in killing animals such as leisure sport, scientists, most likely do not harm animals; if pain is intended on an animal it is strictly for the purpose of scientific advancement. Thirdly, although, animal experimentation may cause some extinction, it is only one of many other causes of extinction, if other causes are not condemned; then neither should animal experiment...
"Polymerase Chain Reaction (PCR) Fact Sheet." National Human Genome Research Institute. 10 Dec. 2007. National Institutes of Health. .
Print. The. The "Polymerase Chain Reaction." Wikipedia. The World of the.
Technology shapes the environment and even food foundations. The technology called genetic manufacturing has shaped the nutrition frugality since the first bacterium to be hereditarily reformed in 1973. There are three classifications used within genetic engineering: the plasmid technique, the vector technique, and the biolistic technique. The plasmid method, frequently the utmost used process includes bacteria providing plasmids, a minuscule sphere of DNA (The Jackson Laboratory). The rings that the plasmids emit are duplicating molecular generators within the cell. Plasmids are essentially indispensable to genetically contrived cells in the wildlife. Plasmids deliver an operational way in which characteristics that are not typically within a chromosome can be conceded from one cell to an alternative cell. Very few plasmids acquire genes that encode for enzymes such as penicillin or ampicillin and these materials dissolve antibiotics permitting a vast subsidy to the cell because they now become invulnerable to numerous classes of antibiotics. When these cells enclosing plasmids ceases from living adjacent cells clutch the plasmids and acclimate to the qualities that were attained in the previous transaction. He...