Nightmare Before Christmas | Analysis

Nightmare Before Christmas | Analysis Tim Burton rings in my ears as one of Hollywoods most eccentric directors. There is a distinct and unique phenomenon in his films: the genre of his most films are between animation films and real person films; his animation films overturn the world of traditional fairy tales, showing the darkness of horror, but did not give us the feel of fear. Nightmare before Christmas, which reflects his typical paradigm, is a fantasy mixed of horror, magic, darkness, holidays, the jolly guys, and an interesting skeleton. The features of Nightmare before Christmas have a similarity with Bakhtins theory of carnivalesque. In Bakhtins view, everyone lives a carnivalistic life as long as those carnival laws. His feel of carnival is full of grotesque realism, which can get rid of the horrable things in the world. And the grotesque style can also turn the world into a bright and cheerful one. (Bakhtin,1989). There is always a feel of carnival surrounded in Nightmare before Christmas. Because of the feel, audience can get away from the fear successfully. On the other hand, Bakhtin believes that carnivalistic life is a life drawing out of its usual rut; it is to some extent life turned inside out, the reverse side of the world.( Bakhtin,1989) Similarly, Burton sets two worlds to make a comparision: the human world and underground world; the grotesque world of Halloween and the happy world of Christmas. The confrontation of the two worlds represents Burtons inclination. The happy world is boring while the eerie wolrd is vivid and interesting. In my Nightmare before Christmas lingers on the edge of conventional culture and popular culture, it challenges the conventional culture and queries on the popular culture. So Burton makes an utmost effort to prove the rationality of his subversion in this film. It takes delight in talking about Burtons Gothic darkness of horror. The story happens in the world of ghosts and monsters where we are totally un familiar. The vampire bats, red eye demon , black monsters and even the wretched green, these gothic images are everywhere in the film. They stand in stark contrast to the simplicity of traditional animation films. However, the audience feel more closer to them and come to realize their subsistence status trenchantly. The American animation films have a heroism complex, but Burton deconstructs our expectation of the traditional hero. In Nightmare before Christmas, the character Jack is frightful and he has no mission to save the world. Other character is treated equally, eg. the pet comes along with Jack is soul of a dog instead of a brilliant white horse. More importantly, Burton wants to create a balance between creepy and interesting. So he gives life to the grotesquery Jack who actually is virtuous. He demonstrates the characters individual emotions and action which has no relationship with hero. Specificly, the modeling of Jacks eyes are just two black holes which thoroughly give expression to the meaning of the character. Lets turn to the other film: No Country for Old Men In this film No Country for Old Men the genre pushes the envelope of sanity. It is an unconventional western film, mixed with action, crime drama and dark humor. The McCarthys novel No Country for Old Men is an ideal vehicle for the Coen brothers, who have used violence and emptiness laced with dark humor as an artistic aesthetic in their directing careers. The films greatest achievement is its ability to transport the audience into a world where the predestination, death, fears, choices, morals and realities of life are strikingly brought to life and make the audience reflect upon and look back with careful consideration after the whole experience. The violence and bleakness of it all is not to simply evoke reaction or engage the audience, it is to tell a story and impart an experience of great intention, to which the Coens have brilliantly succeeded. To some extent, this film is a character study of Sheriff Bell, an honest lawman who is wise, observant, grounded in reality, and has a long memory. No Country for Old Men is really his story. And also, The Coen brothers direction of the particularly intense chase scenes between Chigurgh and Moss are masterful, evoking emotions of suspense to the highest level and pushing the audience to the very edge of their seats. At the same time the movie is a character study on the effects of evil and innocence lost, an exploration on the themes of fate and chance, an analysis of the freedom to choose and its consequences, a reflection on evil and good as forces of society and the investigation of basic human emotions such as hope, fear, love, violence and aspiration in the face of a variety of situations. The ending of the film is about death. Faced with the overwhelming fact of death, all of us humans find meaning only by making up their own spiritual codes. The murderers code, the good old boys code, the sheriffs code, the wifes codethey are all equally meaningless, equally inadequate in the face of death, but they are all perfectly legitimate responses to the fact of universal death. The film is also about predestination and free will, Chigurgh doesnt believe in free will, I got here the same way the coin did , he is almost like a robot , and scientifically he is totally right we dont have free will , Chigurgh is not guilty , he just has killer genes and his mother didnt hug him so thats why he turned out to be this way, there are no choices in matter , just cause and effect , only quantum psychics can show some difference. In conclusion, Film genres can take culture as very important elements into consideration. And also, we can characterize a culture by its genre set. Obviously through the film review, we can see that genre can function as a tool to define and describe a culture. Heroes and Villains: Historical Analysis Heroes and Villains: Historical Analysis Heroes, Villains or Both? Austin Rappel What I found most interesting in this weeks readings was the myths behind the men; specifically Ulysses S. Grant, general in chief of the United States Army, William T. Sherman, general in The United States Army and Abraham Lincoln, president of the United States. Grant was considered a drunk, Whatever happened, the story of his drinking became a staple of gossip in the old army. (McPherson, pg.114) Sherman was considered crazy; But Sherman could never entirely escape the reputation of madness (McPherson, pg 114) Lincoln was considered passive: a basic trait of character evident throughout Lincolns life: the essential passivity of his nature. (McPherson, pg. 206) I think it is important to determine if these things were true or if there is enough evidence to ever know for sure the mindset or character of the men that are known to us as the men that defined and ultimately were the victors of the Civil War. Grant quickly rose through the ranks during the Civil War; command of a brigade, a division, an army (Army of Tennessee), an army group, an all of the armies of the United States. (McPherson, pg 110) These accomplishments are a big contrast to what one could consider could be an accomplished by a drunkard. Brooks Simpson , a biographer concluded, Although Grant sometimes took a drink during the war, and may on occasion have taken two, his colleagues who knew him best and were in the best position to observe him were unanimous in their testimony that he was rarely if ever drunk. (McPherson, pg. 114) Grant was also given the reputation as a butcher when the general most deserving of the title was Robert E. Lee. (McPherson, pg 113) Sherman is best known for his March to the Sea; a march of over 700 miles, in which psychological warfare was used in order to stop the war. As stated in McPherson, The terror that his soldiers provoked among Southern whites was a power, he wrote, and I intended to utilize it to humble their pride, to follow them to their inmost recesses, and to make them fear and dread usà ¢Ã¢â€š ¬Ã‚ ¦ We cannot change the hearts and minds of those people of the South, but we can make war so terrible and make them so sick of war that generations would pass away before they would again appeal to it.(McPherson, pg. 124) As stated in the article by Matt Carr, General Shermans March to the Sea, Shermans plan was to attack the infrastructure of the south and therefore end the war; After more than three years of violent and seemingly endless conflict, Sherman had decided to take the conflict beyond the battlefield and subject Georgia to a level of devastation that would make its population realise that w ar and ruin are synonymous terms. (pg. 30) Its hard to imagine that these strategies were those of a crazy man. Lincoln, the 16th president of the United States and the author of many documents most notably the Emancipation Proclamation was seen at least by one biographer, David Herbert Donald, to be passive. However, as stated in McPherson, at the very outset of his presidency Lincoln demonstrated traits that were the opposite of what Donald calls his essentially passive personality. (McPherson, pg. 207) Its hard to believe that the man that set into motion the Civil War and wrote one of our most historical documents has a passive personality. Maybe the passivity noted by some is more a compassion than anything else. As stated in, Lincolns Legacy, Lincoln Lives Through His Words by Gail Fineberg, Lincolns first draft of the Emancipation Proclamation, which he presented to his cabinet on July 22, 1862. Nobody liked it Sellers said. Lincoln came back to his cabinet with a second draft in September, and admonished members to comment not on substance but on his style. (pg 38) The quote goes witho ut saying to the mindset of a determined man more so to the mindset of a passive man. Although it may never be known the actual character of Grant, Sherman or  Lincoln, it will be left up to the readers and historians perceptions of whether are not these men were heroes, villains or maybe a little of both during and after the Civil War. It goes without saying that these men, however viewed, will always be remembered for the mark they left on the Civil War and wars to come. Polymerase Chain Reaction (PCR): Optimization Parameters Polymerase Chain Reaction (PCR): Optimization Parameters IGHODARO OGHOGHO UYIOSA Polymerase chain reaction (PCR): Evaluation of different optimization parameters for appropriate PCR process. Abstract PCR is a method used to produce relatively large amounts of a specific DNA sequence. The productivity of PCR method depends on different reaction conditions such as the concentration of the DNA template, concentration of magnesium ions, DNA template dilution and polymerase concentration. The aim of this study was to find optimal reaction conditions required for appropriate PCR process. To check the correct conditions the agarose gel and polyacrylamide gel electrophoresis were used. One single, strong amplified band and no unspecific product describe the most suitable amount of given conditions. In the results of this experiment, the most suitable annealing temperature was 62oC, the most proper amount of concentration of magnesium was 2nMol, and the best template dilution was 2 µl. The important parameters evaluated in this experiment were template optimisation, annealing temperatures and magnesium chloride concentrations with and without touchdown PCR. Introduction PCR is a simple, enzymatic assay, which allows for the amplification of a specific DNA fragment from a complex pool of DNA. PCR can be done using source DNA from a variety of tissues and organisms, including peripheral blood, skin, hair, saliva, and microbes. Only trace amounts of DNA are needed for PCR to generate enough copies to be analysed using conventional laboratory methods. For this reason, PCR is a sensitive assay (Lilit andNidhi 2013). To carry out a PCR it is essential to have the following reagents: DNA template, forward and reverse primers, PCR buffer, magnesium chloride (MgCl2), dNTP and DNA Taq polymerase. The annealing temperature allows for the annealing of the primers to the single stranded DNA. It depends on the length and composition of the primers. If the temperature is too high, then the primers will not anneal correctly, and if the annealing temperature is too low then the primers will anneal non-specifically (Hecker et al. 1996). Magnesium ions interact with the DNA polymerase enzyme during this process. The magnesium ion interacts with negatively charged molecules in the reaction. Positive ions of magnesium interact with the negatively charged DNA strands to mask the forces of repulsion (Markoulatos et al. 2002). Template DNA is a fragment of DNA which is needed to create required copies. DNA Taq polymerase is a polymerase enzyme, which is essential for DNA replication, this means that DNA polymerase synthesizes DNA molecules from their nucleotide building blocks (Huang et al. 1992). The nucleotides include the four bases – adenine, thymine, cytosine, and guanine (A, T, C, G) – that are found in DNA. These act as the building blocks that are used by the DNA polymerase to create the resultant PCR product. During this experiment, two methods were used to visualise the PCR products formed. These were Polyacrylamide gel electrophoresis (PAGE) and Agarose gel electrophoresis method. Electrophoresis is a separation procedure which is based on the separation of DNA fragments by size, shape and charge. The clue of this process is the mobility of ions in an electric field (nucleoid acids, which are negatively charged migrate to the anode – positive electrode) (Stellwagen, 1998). Aim The aim of this experiment was to assess which parameters as annealing temperature, concentration of magnesium, and template DNA influence DNA amplification efficiency and specificity. 2.0  Materials and methods This section was divided into two parts. In part A, a PCR reaction 1 set-up using optimised PCR mastermix was done, while in part B, a PCR reaction was set-up to test four different variables to optimise a PCR reaction. 2.1  Part A Setting up the PCR reaction A mastermix enough for four reactions was made. 30 µl of H2O, 50 µl of 2X PCR mastermix and 4 µl each of forward and reverse primers were pipetted and mixed in an Eppendorf tube from which, 24 µl of the mastermix was pipetted into three separate PCR tubes (i.e. tube 1, 2, and 3). 1 µl of sample DNA, 1 µl of 1/10 diluted DNA and 1 µl of H2O were added to each tubes respectively and each amplified on a PCR block running the following programme: Denaturing step done at 94oC for two minutes Amplification step done at 94oC for 30 seconds, 55oC for 30 seconds and 72oC for 1 minute. This step was repeated for 35 cycles. Finally, the extension step was done at 72oC for 3 minutes. Agarose gel electrophoresis (2% agarose gel for PCR) An agarose gel was submerged in a gel tank filled with TBE buffer. Then 5 µl of gel loading buffer was added to each sample and mixed. Next, the first well was loaded with the molecular weight marker and then 10 µl of each sample was loaded into each respective wells. Next, the gel was run for 45 minutes at 80V. Finally, the gel was visualised on the gel documentation system to show separation and migration of the DNA. 2.2  Part B PCR optimisation reaction For this protocol, PCR reactions were set up and individual components of the reactions were varied in other to optimise them as follows: Annealing temperature optimisation 17.75 µl of water, 2.5 µl of 10x reaction buffer, 0.75 µl of Magnesium Chloride (50mMol), 0.5 µl of dNTPs, 1 µl each of forward and reverse primers, 0.5 µl of Taq polymerase and 1 µl of DNA were pipetted into 5 separate tubes (i.e. tube 1, 2, 3, 4, 5) which were then placed on a gradient PCR block, with one tube at each of the following temperature 46oC, 52oC, 55oC, 58oC, and 65oC. Next, the PCR block was set to the following programme: Denaturing step at 94oC for two minutes Amplification step at 94oC for thirty seconds, 46-65oC for thirty seconds, and 72oC for one minute. This stage was repeated for thirty-five cycles. Finally, the extension step was done at 72oC for three minutes. Template optimisation A PCR mastermix containing 13.75 µl of water, 2.5 µl of 10x reaction buffer, 0.75 µl of 50mMol magnesium chloride, 0.5 µl dNTPs, 1 µl each of forward and reverse primer and 0.5 µl of Taq polymerase were pipetted into five tubes. Next, 5, 2, 1, 0.1, and 0.01 µl of template DNA and, 0, 3, 4, 4.9. 4.99 µl of H2O were added to each tubes respectively. Next, the tubes were then amplified on a PCR block using the same cycle parameters as set out in part A. Magnesium concentration A PCR mastermix mastermix containing 16 µl of water, 2.5 µl of 10x reaction buffer, 0.5 µl dNTPs, 1 µl each of forward and reverse primer, 0.5 µl of Taq polymerase and 1 µl of DNA were pipetted into five tubes (i.e. tube 1, 2, 3, 4, and 5). Next, a Mgcl2 dilution was prepared to get a final Mgcl2 mMol of 0.5, 1.5, 2, 3 and 5mMol which were then added to each tubes respectively to give a final volume of 25 µl. Next, the tubes were then amplified on a PCR block using the same cycle parameters as set out in part A. Touchdown PCR A PCR mastermix was prepared using the same variables outlined for magnesium concentration. Then the touchdown PCR programme used was as follows: 94 °C for 3 minutes then 94 °C for 30 seconds, 64 °C for 30 seconds and 72 °C for one minute for three cycles, 94 °C for 30 seconds, 61 °C for 30 seconds, and 72 °C for one minute for three cycles, 94 °C for 30 seconds, 58 °C for 30 seconds, and 72 °C for one minute for three cycles, 94 °C for 30 seconds, 55 °C for 30 seconds, and 72 °C for one minute for three cycles, 94 °C for 30 seconds, 53 °C for 30 seconds, and 72 °C for one minute for thirty cycles, and finally 72 °C for three minutes. Sample preparation and polyacrylamide gel electrophoresis After all the different optimisation protocols, the samples to be loaded into the gel were prepared by adding 5 µl of loading buffer to each PCR reaction and mixed. Next, 5 µl of DNA ladder was pipetted into the first well while 10 µl of sample were pipetted into each designated wells. The gel was then run at 100V for 45 minutes. After which, the gel was recovered and placed in a weighing boat containing 1x TBE buffer. Next, 5 µl of ethidium bromide was carefully introduced into the weighing boat and left for 10 minutes before visualisation with a gel documentation system. 3.0  Result PCR reactions were set up in 5 different PCR tubes with all the required components for complete PCR reaction , save for a particular factor which was varied to ascertain the optimal concentration necessary for the production of the highest amount of pure specific product. The results obtained from these various optimised factors are represented in the gels below. Part A Optimized PCR 1 µl of DNA template was loaded in the well labelled neat DNA and it revealed the highest amount of product formed, the well containing a 1/10 diluted DNA had a much lower amount of product formed while the well with the water blank yielded no product. Also, unspecific products were not formed. Figure 1: Optimised PCR agarose gel. Part B The results of different PCR optimisation reactions PCR annealing temperature optimisation Figure 2: PCR annealing temperature optimisation polyacrylamide gel. Key: L-molecular weight ladder, lane 1- 48 °c, lane 2- 52 °c, lane 3- 55 °c, lane 4- 62 °c, lane 5- 65 °c, unspecific product, specific product. From Figure 2 above, there are three unspecific products formed in lane 1, a greater specific product with insignificant unspecific product formed in lane 2, insignificant unspecific products formed in lane 3 and there is also a decrease in the intensity of the specific product formed compared with that of lane 2. While in lane 4, there is a minor decrease in the intensity of the specific product formed thus, the intense quantity of the specific product formed (i.e. there is a strong amplification here) and in lane 5, there is a major decrease in the quantity of specific product formed. Therefore, the quantity of the product decreases as the quantity of the template DNA decreases, and the quantity of the specific and unspecific products increases as the quantity of the template DNA increase. Template dilution optimisation Figure 3: Template dilution optimisation polyacrylamide gel. Key: L -molecular weight ladder, lane 1- 5 µl, lane 2- 2 µl, lane 3- 1 µl, lane 4- 0.1 µl, lane 5- 0.01 µl template DNA, specific product, unspecific product. From the gel above, there are three unspecific products formed in lane 1 and also the intensity of the specific product formed is high, the unspecific product formed in lane 2 is insignificant and the specific product formed is greater in intensity (i.e. has a higher amplification) compared with that of lane 1, in lane 3 and 4, the intensity of the specific product formed decreased compared with that of lane 2, while in lane 5, there is a major decrease in the intensity of the specific product formed. Therefore, the quantity of the product decreases as the quantity of the template DNA decreases, and the quantity of the specific and unspecific products increases as the quantity of the template DNA increase. Magnesium dilution optimisation Figure 4: Magnesium dilution optimisation polyacrylamide gel. Key: L -molecular weight ladder, lane 1- 0.5mmol, lane 2- 1.5mmol, lane 3- 2mmol, lane 4- 3mmol, lane 5- 5mmol, specific product, unspecific product. Form the gel above, there is an unspecific and a specific product formed in lane 1, in lane 2 there is a decrease in the intensity of the unspecific product formed and also there is an increase in the amount of specific product formed. In lane 3, there were no production of unspecific products and the intensity of the specific product formed remained high. In lane 4, there is evidence of the presence of an unspecific product formed but the intensity of the specific product formed remained high. While in lane 5, there is a minor reduction in the intensity of the specific product formed while there is visible presence of formation of unspecific products. Magnesium touchdown optimisation Figure 5: –Touchdown magnesium concentration optimisation. Key: L – molecular weight ladder, lane 1- 0.5 mMol, lane 2- 1.5 mMol, lane 3- 2mMol, lane 4- 3 mMol, lane 5- 5mMol, unspecific product, specific product. From the gel above, in lane 1, the intensity of the unspecific product formed is the same with that of the specific product formed. While in lane 2, 3 and 4, the intensity of the specific products formed are the same while the intensity of the unspecific products gradually decreased. There was no unspecific product formed in lane 5, however there was a reduction in the intensity of the specific product formed compared to that of lane 4. Discussion This experiment was performed to evaluate different optimisation protocols to optimise PCR reactions. For the PCR reaction using an optimised PCR mastermix in part A, the highest amount of products formed was observed in the well containing 1 µl of DNA template.The annealing temperature is the most important optimisation, because it can have an influence on the specificity of the reaction. If the temperature is too high, the hybridization will not take place thus, templates and primers remain dissociated. If the temperature is too low, mismatched hybrids will occur. Correct annealing temperature must be low enough to start hybridization between template and primer, and also high enough to prevent forming mismatched hybrids (Roux, 2009). According to the results from annealing temperature optimization polyacrylamide gel (Figure 2), the most suitable annealing temperature was 62oC, because the band was clear and single as opposed to the 48oC, 52oC and 55oC, where the smears (i.e. unspecific products) were shown. The intensity of the band in 62oC was the strongest compared with that o f 65oC. The most suitable template dilution for PCR was 2 µl, because it gave in the polyacrylamide gel in Figure 3, the most bright, single band with very low amount of unspecific products formed. Besides, annealing temperature and template dilution parameters, PCR reaction components could also lead to non-specific amplification. Two variables, which are reported to greatly influence the specificity of the PCR reaction, are magnesium and dNTP concentration (Dwivedi et al. 2003). For magnesium dilution touchdown (Figure 5), the molarities at 1.5, 2 and 3mMol showed very similar amount of products formed indicating the importance of the magnesium in PCR amplification while for magnesium dilution without touchdown (Figure 4), it was found that 2mM yielded the best results. Magnesium concentration is known to play a critical role in amplification as it can affect DNA strand denaturation, primer annealing specificity and enzyme fidelity. These observations are in agreement with earlier studies (Innis et al. 1990; Eeles et al. 1993). Even brief incubations of a PCR mix at temperatures significantly below theTmcan result in primer-dimer formation and nonspecific priming. Hot-start PCR methods (Erlich et al. 1991;Ruano et al. 1992) can dramatically reduce these problems. In this experiment, two methods were used to visualise the PCR products formed. They are, Polyacrylamide gel electrophoresis (PAGE) and Agarose gel electrophoresis. Agarose gel is the most popular medium for the separation of moderate and large-sized nucleic acids and have a wide range of separation but a low resolving power, since the bands formed in the gels tend to be indistinct and spread apart. This is a result of pore size and cannot be largely controlled (Stellwagen, 1998). However, Polyacrylamide gels are normally more difficult to prepare and handle, and it requires a longer time for preparation than agarose gels. However, polyacrylamide gels have a greater resolving power, can accommodate larger quantities of DNA without any significant loss in resolution and the DNA obtained from polyacrylamide gels is extremely pure (Guilliatt, 2002). Hence, they are better than agarose gels. It should be noted that polyacrylamide is a neurotoxin (when unpolymerized), but with proper labo ratory care it is no more dangerous than various commonly used chemicals in the laboratory (Budowle Allen, 1991). Conclusion Optimisation of Polymerase Chain Reaction is very important for PCR performance to minimize failures, avoid the production of non-specific products and increase specificity of the reaction. The knowledge of proper conditions allows to use PCR correctly and to receive good results. According to the results of the research the most suitable annealing temperature was 62oC, the most proper amount of concentration of magnesium was 2nMol, and the best template dilution was 2 µl. Those parameters give DNA amplification specificity and efficiency. (Harris and Jones, 1997). References Alka, D., Sarin, B., Mittar, D., Sehajpal, P. (2003). OPTIMIZATION OF 38 kDa BASED PCR ASSAY FOR DETECTION OF MYCOBACTERIUM TUBERCULOSIS FROM CLINICAL SAMPLES. 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