Compare and contrast Marxist and Functionalist accounts of religion

Both functionalists and Marxists share the common view that religion serves to legitimise the morals and laws within society. Many functionalists as well as Marxists do agree that society creates religion as a visual symbol of itself. Followers are ultimately not worshipping their religion, their worshipping society and everything it stands for. However, this is where the split in views begins. Functionalists see Religion as serving towards the 4 pre-requisites of society. (Namely: Social integration, shared values, social solidarity, and social harmony). By ensuring these needs are met, religion reinforces collective values and promotes solidarity. Functionalism is not over concerned about the ‘why' religion is allowed to assume this falsified symbolism, but instead recognises it's use in keeping harmony and faith in society. Durkheim demonstrates this in admitting that religion does not have to be ‘super natural'. It's important to note that mortal people and objects can gain a sacred status in society similar to that of the idols of religion. For instance: Princess Diana – a national symbol of charity and possibly the last real ‘princess' of the British Nation in the people's hearts. She has become sacred because she represents a good quality of society. From this, Durkheim believes â€Å"Understand what sacred things represent and you understand the values of a society†. Here we come into the first major difference between functionalism and Marxism. The former believes that there is a positive relationship between society and the individual. Worship society; believe in society. Marxists, however, inherently are discontent and have an ill view of society as it is. It is understandable how they are opposed to religion indoctrinating the people into a value set, pulling them into the collective conscience that ultimately supports capitalism and the idea that people deserve to â€Å"have their place†. Religion has allows supported class systems and promoted inequality. In gender. In work. In authority. Everything that Marxism is opposed to. â€Å"The rich man placed in his castle – The poor man placed at his gate† Malinowski, famed for his research at the Trabaind islands, did not see religion as a celebration of society, despite the same functionalist perspective as Durkheim. He did, however, agree that religion promotes solidarity. It does so by dealing with emotional stress / life crisis (disruptive events). Religion goes as far as to introduce ceremonies for dealing with various life crisis. Death is given a funeral. Love is given marriage. In all cases then hope is given through the expressed belief in immortality and fellow mourners serve to comfort and support the bereaved, so they can become functional members of society once again. Dangerous and unpredictable events are also surrounded in religious ceremony. Prayer is common before a possibly hazardous experience. These rituals reduce anxiety and increase confidence, strengthening unity in shared situations. Talcott Parsons shares this view and goes onto show how religious devices, such as the 10 commandments, provide the basis for many social norms and morals. Religion guides behaviour and helps in the formulation of decision through this. Finally, Religion is looked to answer the â€Å"ultimate questions† and give meaning to our existence. Humanity needs to feel as though there is meaning in all significant things; meaning to death and suffering, and justification of existence in itself. Religion works to show sense in contradictory scenarios that threaten the balance of ultimate beliefs. For instance, a man who is profiting through the doing of evil will be punished in the after life for his wrongful gain in life. Religion is a warning, a giver of justice, a provider of guidance and ultimate supernatural fear if one goes astray. Marx himself rejects any idea of supernatural aspect of religion. He describes religion as the â€Å"Heart of a heartless world†; The heartless world being the society moulded by capitalism. Religion is born out of a need for comfort, it does just this by cushioning the effect of worker oppression. Marxists have placed religion as part of the super structure, it helps justify the capitalist base as well as the whole super structure. (See rich man quote page 1). Religion as a tool of the bourgeoisie plays an important roll in justifying worker oppression, by showing that all will gain equal reward in heaven for doing the ‘right thing'. Religion has varying levels of strictness according to socio-economic class placement, especially in India where the cast system places strict restrictions and categorisations on a persons place in society. The idea of equal opportunity is ultimately crushed by harsh teachings and acceptance that a lower class worker is having a bad life because super natural forces placed them in that situation as punishment for wrongfulness in previous life. In conclusion, Marxist and functionalist similarities on the subject start and end with the acceptance of religion as a conservative force in society. Neo Marxism does accept the idea (like functionalism) that religion can sometimes be useful to society in bringing about change for the better. For instance the radical role of Liberation Theology. (Madura). Traditional Marxism is totally opposed to the oppressive role of religion and would be surprised to see that radical forces have emerged with some minority religious groups. Functionalists such as Durkheim and Parsons see religion as being a positive and perhaps essential part of the harmonious workings of society but have been criticised for ignoring the dysfunctional, disruptive, and divisive aspects of religion. They fail to consider hostility between religious groups within the same society. â€Å"It would seem that religion threatens social integration as readily as it contributes to it† (Stark & Glock).

Representations of Love in Much Ado About Nothing

Explore representations of love in Much Ado About Nothing In Much Ado About Nothing, Shakespeare uses literary structures such as doubles and opposites in order to emphasise the plays main themes and ideas. McEachern claims â€Å"It is undoubtedly the most socially and psychologically realistic of his comedies, in it’s portrait of the foibles generosities of communal life. † (McEachern, 2006, 1) One main theme I want to explore is love and how Shakespeare represents this in Much Ado About Nothing. The two main genres in Shakespeare’s dramas are tragedy and comedy. Tragedy always ends in death and comedy always ends in a marriage. Although Much Ado inevitably ends in marriage, it differs from some of Shakespeare’s other romantic comedies as his other comedies usually portray love in a much more unrealistic way. â€Å"Much Ado About Nothing is best known for the ‘merry war’ between one of it’s two couples, and an oxymoron could also describe this comedy’s identity as a whole. Shakespeare offers a play of light and dark, of romantic union wrested from fear and malice and of social harmony soothing the savagery of psychic violence† (McEachern, 2006, 1) In Act 1 Scene 1, Don Pedro, prince of Arragon arrives with his bastard brother Don John, and his two friends Claudio and Benedick. It is in this Act that Beatrice and Benedick first meet and the war of wits begin. Leonato states â€Å"There is a kind of merry war betwixt Signior Benedick and her [Beatrice]; they never meet but there is a skirmish of wit between them. † (Much Ado, Act 1 Scene 1, 520) Although their words seem quite hateful to one another, at the same time it may also come across as flirtatious. Before Beatrice even meets Benedick, she expresses her distaste for him, however, she talks about him in such great depth it is almost more like an obsession than hatred. Benedick teases Beatrice by saying â€Å"I am loved of all ladies, only you excepted: and I would I could find it in my heart that I had not a hard heart; for, truly, I love none. † (Act 1 scene 1, 521) to which Beatrice replies â€Å"A dear happiness to women: they would else have been troubled with a pernicious suitor. I thank God and my cold blood, I am of your humour for that: I had rather hear my dog bark at a crow than a man swear he loves me† (Act 1 Scene 1, 521) By using the characters of Beatrice and Benedick, Shakespeare mocks the conventional type of love, romantic love, which is expressed by Hero and Claudio. This is also an example of how Shakespeare uses doubles in his play, as he uses the two couples to express two types of love. One being more realistic, that of Beatrice and Benedick, and the other being the more unrealistic, over the top romantic love expressed by Hero and Claudio. Even though the plot is largely based on Hero and Claudio’s relationship, the witty banter and seemingly unromantic relationship between Beatrice and Benedick seems much more interesting to the reader and we are more interested in how their relationship will develop. It is this relationship that seems much more believable compared to Claudio and Hero’s fairytale love at first sight. It is through contrasting these two different types of love and through the different use of language that Shakespeare can mock the conventional romantic love. Claudio uses a totally different style of language to Benedick when they both speak of love. Benedick is highly cynical and negative about love where as Claudio is more pretentious and elaborate when he speaks about Hero, for example when he says â€Å"Can the world buy such a Jewel? † (Act 1 scene 1, 522). This language is completely different to the way that benedick speaks to Beatrice as the first thing he says to her is â€Å"My dear Lady disdain! Are you yet living? † (Act 1 scene 1, 521) Benedick also speaks of his frustration of Claudio’s eloquent language when speaking of love as he states â€Å"He was wont speak plain and to the purpose, like an honest man and a soldier†¦his words are a very fantastical banquet, just so many strange dishes. (Act 1 Scene 3, 529) This however is quite hypocritical of him as in Act 4 Scene 1, Benedick confesses his love for Beatrice and states â€Å"I do love nothing in the world so well as you: is that not strange? † (Act 4, Scene 1, 541) to which Beatrice responds â€Å"I love you with so much of my heart, that none is left to protest† (Act 4, scene 1, 541) This shows how dramatically their relationship has changed as the witty banter and insults have turned into confessions of love for one another. It is often difficult to understand and accept the love between Hero and Claudio as it is so unrealistic. They fall in love with each other before they even truly get to know one-another, which therefore makes their love and marriage seem quite false and shallow. The fact that Claudio does not question Don John when he professes that Hero has been unfaithful, yet instead believes his word to be true, questions whether the love he has for Hero is sincere. Surely Claudio would confront his future wife before coming to any sort of conclusion, however, even her own father believes this to be true and states â€Å"why she, oh she is fallen/ into a pit of ink, that the wide sea/ hath drops too few to wash her clean again,/ and salt too little, which may season give/ to her foul tainted flash† (Act 4 Scene 1, 540) Another aspect of the play that makes Hero and Claudio’s love very unrealistic is hero’s willingness to forgive Claudio after his bold accusations of her infidelity. If his love for her was as strong and powerful as he made out, he would be more trusting of Hero in the first place. However, she seems to disregard this and does not question his behaviour, but instead is willing to carry on with the marriage. Bibliography McEachern,  Claire. Much Ado About Nothing. 2006. the Arden Shakespeare Shakespeare, W. The Complete Works of William Shakespeare. 1996. Wordsworth Editions Limited

Psy 600

1 Running head: PEER REVIEWED ARTICLE SUMMARY Peer reviewed article summaries PSYCH/600 Developmental Psychology March, 11, 2013 2 Running head: PEER REVIEWED ARTICLE SUMMARY Peer review article summaries Many peer reviewed articles were interesting to me; however the three that really stood out in mind is socioeconomic status and child development, Psychology and Aging: The first 20 years, and Child development. These journals stood out I taught about how it could relate to my live and adolescents at my job.Psychology and aging: The first 20 years speaks about how the aging process through the years of life has change and a few traits are still actively the same as the years went by. One characteristic that remains the same is representation of the broad spectrum of research on the development and aging. Another further type of diversity in Psychology and aging that was mention is the geographical diversity of the how the authors reviewers. Another history of the psychology and agin g process has strived to be broadly inclusive with respect to specific domains of adult development and aging process.Theoretical orientation is applied thrust of the work methodology. Early issues spoke about the basic aspects of cognition, perception, and memory. Socioeconomic status and child development talked about the differences of high and low in families. Through this article it speaks on how children from low-SES families are more likely to experience some form of growth dysfunction. It also shared how children from low –SES families were born prematurely and with some sort of birth defects. 3 Running head: PEER REVIEWED ARTICLE SUMMARYCognitive and academic attainment relationship between SES and low families’ studies has shown that children who come from poverty and low parental education are associated with lower levels of school achievement and IQ later in childhood. As for the highest SES group was shown 25 points higher than the mean of the lowest SES g roup. The link between SES and children’s social and emotional well-being is not always consistent as to cognitive attainment. There is substantial evidence that low- SES children more often manifest symptoms.Another peer reviewed article is socioeconomic status and child development, this topic remains to be interesting it speaks on child development and how children well-being is represents in three domains. Cognitive the intellectual and academic competence and how the child learning behaviors develop as they grow into an adult. Socioemotional development speaks about the social and emotional well-being is not as consistent as the cognitive attainment. The low-SES children more manifest symptoms of psychiatric disturbance and social functioning.The strength of the relationship between SES and mental disorders varies by type of disorders and race. In summary these three articles all share the information and studies that I found relatively interesting. 4 Running head: PEER REVIEWED ARTICLE SUMMARY References: Bradley, R. , & Corwyn, R. F. (2002). Socioeconomic status and child development. , (), Rose, T. Z. , Haley, W. E. , & Blanchard-Fields, F. (2006). Psychology and Aging: The first 20 years. , (), Bradley, R. H. , & Corwyn, R. (2002). Socioeconomic Status and child development. , (), .

Political Economy of Race, Class and Gender Research Paper

Political Economy of Race, Class and Gender - Research Paper Example An increase in population still kept African-Americans and the whites in separate neighborhoods. These separate neighborhoods deprived African-Americans of receiving quality education and competing with the whites in the labor market. Segregation also created a feeling of hostility between the two communities who still fail to interact and comprehend the behavior of each other. This research paper will investigate the ways in which segregation among different communities, namely African American and the whites, creates inequality in the labor market. For this reason, this paper will examine three very important elements to understand why the problem of income disparity exists in one of the largest metropolitan cities of the USA. The three important elements identified by research are demographics, education and unemployment. Demographics play a critical role in understanding the labor market of Chicago. Chicago is one of the densely populated cities of the US with  a population of over 2.5 million people out of which about 32% are African Americans while 45% are white (Reich 17). However, Chicago is highly segregated. The major ethnic groups like to live among themselves in what they call their neighborhood. Jobs are highly dependent upon the neighborhood where people live. In order to understand the labor market of Chicago, it is critical to look at some of the details in history. The Second World War gave opportunity for the masses to work in factories as the US prepared for war. The Chicago Defender broke the news to African-Americans living in the South that there were plenty of jobs in the city of Chicago and ensured African-Americans would live a much happier life there (Ellen 261-277). It worked for most private firms, who were looking for cheap labor and also for Africans American, who found it difficult to find jobs at that time. There were nearly 3,000 African Americans

Team Work Essay Example | Topics and Well Written Essays - 250 words - 1

Team Work - Essay Example ly with project team members in the form of knowing how projects shape up, where resources are being utilized, how the social styles play an imperative role at telling the team members about their linkages and development routines, and how their overall linkage dictates the negativities and issues which may arise in the future. These dimensions present within the social styles model aim to oversee any critical areas which may need immediate attention, a comprehension to set things right and an overall feel to get the job done for the basis of the teams that have been lined up. It is a fact that communication may be tailored in such a way to accommodate the communication styles of the people that are based on the dimensions of the social styles phenomenon. This is because when social linkages get developed, there are mannerisms which will take care of the work processes and how their internal relationships will pave the way for growth and development across the board. This is an important precedent for resolving the predicaments related with the social styles

Civil Partnership, Marriage & Cohabitation Essay

Civil Partnership, Marriage & Cohabitation - Essay Example Standard property law is used for these couples.2 Cohabitation law may extend to opposite sex couples, same-sex couples, and platonic pairs.3 Therefore, just like with standard property law, if a cohabiting couple breaks up, and it is shown that one of the partners has not financially contributed to the home, even if that partner has contributed child care or labor, then than that partner is still not afforded an interest in the property. This is different in marriage – in marriage, the property is divided equitably between the spouses, regardless of the amount of financial contribution from the parties, or the lack thereof.4 This essay will examine the issues surrounding people who cohabit, as well as the specific cases regarding cohabitation, and will attempt to make recommendations that would help couples be better protected under the UK law. Cohabitation Law verses Marriage Law Property division is an issue when considering cohabitation law and marriage law. Cohabiting cou ples do not have the same property rights as married couples.5 Basically, if there is a cohabiting couple, and they break up, their property would be divided according to ordinary property law, which basically means that the property follows the title – if, for instance, there is a house with only one person's name on the deed, then that person alone is entitled to the property. If both person's name is on the deed, then the property would be divided between them evenly.6 With marital law, it is different – if a marital couple divorces, the property is divided between them equitably, regardless of whose name is on the deed.7 The Marital Causes Act 1973 gives a court the right to order the transfer of property from one party to the other upon the filing of a dissolution of marriage, or the court may also order the sale of property as well.8 This principle is shown in several cases that have been decided in UK Courts. One is Burns v. Burns, which states that if there is a non-working partner in a cohabiting relationship, and that non-working partner does not contribute financially towards the purchase of the house, or the mortgage payments, than that spouse is not entitled to a division of that property.9 The plaintiff in Burns lived with the defendant, without the benefit of marriage. Valerie, the plaintiff in Burns, took care of the children and stayed home, forgoing a career and money of her own. That said, when the relationship progressed Valerie went to work, and paid domestic bills from her salary, along with buying furniture and equipment for the house, while also spending money on children's clothes. She also decorated the interior of the house.10 Despite all that the plaintiff contributed to the household, the plaintiff did not get property rights in the house after the couple broke up. This was because Valerie's contribution to the household did not directly contribute to the finances of the home- she neither made house payments, nor did she contribute to the down-payment of the home.11 This principle was echoed in Lloyds Bank plc v. Rosset, which is a case involving a married couple. 12 In the Lloyds case, the court held that only financial contributions to the home, not conduct alone, would be relevant in awarding property rights, therefore the partner who did not contribute financially was not entitled to any portion of the house. Abbott v. Abbott,13

Six Sigma Term Paper Example | Topics and Well Written Essays - 2500 words

Six Sigma - Term Paper Example In this respect, a manufacturer produces products that are of the same quality standards and which do not have defects that could lender them unusable and inefficient in performance of the intended services. Conclusively, Six Sigma enables manufacturers to produce products that are within a level of quality that is not extremely different from each other so long as they have a similar brand name. Introduction In the initial implementation of Six Sigma, the aspects that manufacturers considered were focused on the defects and products without defects. These manufacturers set some platform for measuring the standards according to outlined parameters. With Six Sigma, many manufacturers were able to establish parameters to moderate their production of commodities in the market so that the products were able to compare to the earlier produced having similar brand name. However, with time, some parameters that were related to time were developed to measure quality in the process of product ion so as to indicate the importance of efficiency. ... This means that efficiency in production is an aspect that is essential to measure the quality of production that could be experienced in any manufacturing company because it plays an essential role (Eckes, 2003, p53). Due to the numerous benefits associated with this management approach, its acceptance by various fields/organizations s been positive. This has been due to the fact that it effectively addressed problems experienced in the use of traditional management approaches. Other aspects of the Six Sigma included customer satisfaction, reduction in cost, increase in profit and reducing the pollution effects resulting from the use of a product. According to these values, people ensure that they improve the quality of production by ensuring that they are no issues/properties that cause defects while maintaining lowest levels of variability in manufacturing and business processes. This ensures that all people using the strategy are able to moderate the various things in the process of production so that they can ensure that they can sustain high quality or raise it, which would be beneficial to the company in terms of the sales level. The process uses some aspects of quality management to ensure that all the people are able to analyze the process of production so that it is acceptable in production. In this aspect, various things are put into consideration in the course of production to ensure that production has met some quality standards and are within some standards of production. On the other hand, according to the standards used in Six Sigma strategy, the producers are categorized in relation to their level of expertise on the production process (Schonberger, 2008, p.15). Aspects of Six Sigma In the

Life Cycle Nutrition Lab Report Example | Topics and Well Written Essays - 250 words

Life Cycle Nutrition - Lab Report Example This experiment was helpful in acquiring dietary information necessary to maintain a healthy body. The second lab dealt with enzyme and lactose intolerance. It was conducted to determine the ability of Lactaid to digest lactose. It was proven that Lactaid is effective when combined with milk and acid. Academic and scientific integrity should also be taken into account in the field of nutritional science. Consumers are entitled to truthful nutritional information. Otherwise, their health may be compromised. These experiments are beneficial in gaining more knowledge and awareness on our regular food intake. It encourages us to carefully select our food and evaluate our medications. As much as possible, efforts should be made to separate food myths from facts. In relation to the chosen Public Service Announcement (see Part III), our food is our responsibility. Therefore, the value of healthy food consumption among Americans should never be undermined. Ideally, food preference should not be based on availability alone, but more importantly on nutritional

RM Assessment 1 - Critical Revise (marketing) Essay

RM Assessment 1 - Critical Revise (marketing) - Essay Example This report critically reviews the Bansal and Voyer article. The current trend in WOM advertising and marketing includes ethically questionable tactics such as viral videos, guerrilla marketing and paid endorsements of email or Facebook friends and family, or celebrity pages. Litven, etal proposed this modern definition, â€Å"WOM is the communication between consumers about a product, service, or a company in which the sources are considered independent of commercial influence†. (2005) This statement implies the perceived independence is actually more important than the actual independence. The Litven, etal. Article considers travel and hospitality business and the attempt to capture positive WOM. Since many people consider travel risky in terms of settling onto a poor choice of destination or accommodation, travelers seek WOM advice from prior journeys. The internet has become a resource for destination information, some independent, some apparently independent. Email, websites and blogs are asynchronous communications that control the outgoing message. The travel industry as well as other high risk applications should indulge in this form of WOM. Pictures still tell a story better than words, and corporate WOM marketing should include photographic evidence, especially to combat negative WOM. The Lee, etal. article quantifies the effects of WOM on business bottom lines. This study demonstrates the long-term lifetime value of WOM as opposed to indirect marketing. WOM is shown to create a definite value. (2006) The Villanueva, etal study relates closely to the Lee article; however, Villanueva looks at acquisition costs and equity return of building clientele by mass marketing and WOM. WOM proves to be more cost effective, although slower, than mass marketing because WOM provides the best clientele. Long-term customers come from WOM whereas one time customers, the wrong customers, come from mass marketing efforts. (2006) A number of features of the

Project management Essay Example | Topics and Well Written Essays - 3750 words - 1

Project management - Essay Example it is not always the case that these projects are successful and achieve the desired objectives, but usually most of them do fail, either in terms of cost, quality, deliverables or time (Meredith & Mantel, 2012). Any project’s success is inherited in the timely ending of the project, attainment of the required objectives or deliverables are delivered, and within the project limitations or constraints. In order to execute the project successfully, it is imperative for the project team or managers to consider some crucial elements of the project, which may be the project cost, time, or resources (Oisen, 1971; Lock, 1994; Atkinson, 1999). Among these aspects, there are three central issues, such as the time, cost and quality, which are the key intentions of any project and hardly be achieved and make the project a successful one (Iqbal, Azam, & Qureshi, 2011). However, the secret of success can be understood by analyzing the executed projects, where the success factors or failure factors can be identified by a careful analysis. The purpose of this paper is to analyze the two real-life projects related to the adoption of Information Technology in the organization by identifying the success factors of these projects, and also to identify the risk factors with the help of risk map as well as provide suggestions for mitigating or managing the risks. ECourier is a well-known UK based organization, where it provides 24/7 and same day courier services to the customers throughout UK. It was started in 2003 and the main objective of the company was to deliver courier service with accurate delivery information or transparency in this information, along with the highest customer relationships through automation. It collects and delivers the packages from all the areas in London, as per customer’s instructions. In the beginning, it was involved in delivering the packages only in London, but now it has developed a huge customer base across the world, and have built many

Adult learning and motivation Essay Example for Free

Adult learning and motivation Essay An exploration in to the multi-dimensionality of participatory behaviour; and what motivates adults to return to education. The research question that was initially formulated aimed to inductively generate a theory (Rothchild 2006; Cohen et al., 2000). Unfortunately, the initial question became subject to ethical challenges; and within a framework that would demonstrate rigour, validity and reliability, unsurprisingly; it was far better too approach/explore the multi-dimensionality of participatory behaviour; and what motivates adults to return to education. An enquiry designed around this approach has a better fit to a pragmatic framework (Armitage and Keeble-Allen, 2007; Bryman, 2007) and the BERA ethical directives. Furthermore, this should help bolster the totality of coherence; or as Moss et al., (2009) would suggest as; ‘a chain of reasoning and logic’. Similarly, individual agency and how the experiences of adult learners’ are co/re-constructed (Clark 2011; Flowers 2009; p. 3) needed a greater relationship to an interpretivist epistemology (E891 Part 2: Action 2.9; Gage 1989). As the researcher primarily overlooked these factors that, in turn, determine what is seen as valid and invalid knowledge; then [those] factors would have been overlooked when inferences were made during the research process reducing the quality and internal and possibly external validity. Obviously, this incommensurability will be addressed before the researcher analyses any data generated (Bryman, 2007; p. 19). With these approaches better placed the researcher could demonstrate that generally social and cognitive phenomena are simultaneously quantitative and qualitative (Ercikan and Roth, 2006; p.16) and participatory behaviour is an outcome of the ‘meaning-made’ (Clark 2011) i.e. social-cognitive collocation. This would then show that cognition is co-constructed (Clark 2011) and re-constructed by experience resulting in the multiple interpretations that create the social realities in which people act (Flowers 2009; p. 3). It could be suggested that the initial ‘meaning-made’ is a primary motivator which persists until the time the learner feels satisfied (Park and Choi 2009), or, has achieved ‘what they set out to achieve’ (Gustafsson Mouwitz, 2008). This also implies that ‘meaning-made’ is mutable (Gibbons Bylsma 1984) and subject to further co/re-construction; adjustment; or complete abandonment. Research philosophy After extensive ‘Adult learner’ research and talking with tutors that instruct adult learners’ highlighted a distinct difference in the approaches from which children (Pedagogy – teaching method) and adults (Andragogy – teach how to learn) are taught. The implementation of informal learning methods, however, appears to have dominance in the adult education field (Gibbons Bylsma 1984). Therefore, in order to shape and advance the theory, research design and instrument/s required conducting a focussed literature review of several learning theories (see fig 1); namely, Knowles’s Andragogy Theory (Houde 2006), Cross’s Characteristics of Adult Learners (CAL) (Kohl-Frey and Schmid-Ruhe 2007; Crittenton Women’s Union 2012), Margin (Gibbons Bylsma, 1984) and Proficiency Theory (Gustafsson Mouwitz, 2008). Fig. 1 is showing the associated theories that characterize adult learners’ What becomes problematic is; adult learning has not been researched as vigorously as others areas of education, so the real challenge will be – as Hodkinson and Macleod (2010) encountered to anchor the line of enquiry in a combined paradigmatic harbor. In contrast to Hodkinson and Macleod (2010), the upcoming report will be combining the aforementioned theories with the following paradigms’ as they display a distinct homogeneity. Specifically, social (E891 Part 2: Action 2.5), and cognitive constructionism (De Abreu 2000), Interpretivism (E891 Part 2: Action 2.4; Gage 1989) with quantitative and qualitative data collection i.e. mixed methodology. A critical review of the initial report by Street (2013) and Holmes (2013) exemplified the scarcity of knowledge and understanding some had on the associated theories. Both commented on differing aspects of the line of enquiry, but these were conceptual in nature. Street (2013) illustrated that the researcher must remain aware of the macro/micro societal effect that the learning environment has on the adults lived/shared experience and Holmes (2013) suggested that there needed to be a better fit to the realities of the adult learner. With this in mind I re-conceptualized the report and reflected more specifically on the feedback and guidance. Therefore, in order to steer the paradigms so that they pull in the same direction, the aforementioned theories naturally occurring and overlapping dimensions will be grouped (i.e. constant comparison method; Cohen et al., 2000; p. 151) by their substantive statements (i.e. content analysis; Gillham, 2000; p. 137) and used to engender questions. This process generated four themes that naturally expanded upon their shared features. Social contact and Relationships Goal and relevancy orientated External expectations Internal expectations In order to check for consistencies/inconsistencies (Denscombe, 1999; p. 217-8) between the questionnaires i.e. Phase 1 and Phase 2 and interview responses both datasets will be triangulated to assess the overall motivation/s toward participatory behaviour i.e. cross-sectional design (Bryman, 2006; p. 104). This ‘Mutual’ approach (Armitage and Keeble-Allen, 2007) will be implemented during the adult learners’ regular session/s, which should (1) reduce bias (Nederhof, 1985) and attrition (Torgerson 2009), (2) be more pragmatic than experimental research (Torgerson 2009), (3) increase internal validity, reliability and research quality, (4) support external validity and (5) decrease demand characteristics due to any researcher effects. Research enquiries can be polarized into qualitative and quantitative classifications based on how phenomena are represented (Ercikan and Roth, 2006). But, the researcher firmly believes; if representative qualitative and quantitative data have shared aspects that are dependent on their counterpart for completeness (Ercikan and Roth, 2006; p.16; Bryman, 2006; Bryman, 2007), then the incorporation of cross-validation is warranted to best serve this enquiry. This strategy should ensure internal validity; especially when considering using complementary methods (Armitage and Keeble-Allen, 2007). Moreover, as these quantitative and qualitative counterparts contain a fundamental element of the interactive dependency that is shared, and required, for individual understanding i.e. the connectivity of interactivity and the influence on representative individuality then the research must be aware to consider that both methods have shared and conflicting elements. Consequently, when considering multidisciplinary approaches, mixed methods i.e. quantitative and qualitative and triangulation one must be aware that incommensurability can exist between them. Brannen (2005) suggests that some methods become more feasible than others and deemed a better ‘fit’ as [they] provide more sensitivity when investigating complex social phenomena. Hence, certain methods, used in conjunction can become less than complimentary with the other. Additionally, Yin (2006) suggests that the ability to tighten the use of mixed methods so that they do in fact occur as part of a single study requires integration. The claim is that, the more that a single study integrates mixed methods, the more that mixed methods research, as opposed to multiple studies, is taking place (Yin, 2006). Furthermore, Houghton et al., (2010) highlight one of the ethical challenges, which have important implications for qualitative research, practical examples and solutions. The unpredictability of qualitative research means that an a priori prescription for ethical conduct is not always possible. Therefore, the researcher must be constantly mindful of the on-going impact that the research might have on those involved, while simultaneously being ethically sensitive and morally competent Although, mixing methods does provide an inferential narrative to the statistical outputs from quantitative analysis, it might not sufficiently negate the qualitative and quantitative dichotomy (Yin, 2006), or, necessarily produce the expected scholarly standard for presenting credible evidence (Maclure, 2005). These qualitative and quantitative complements are noticeably even arguably intrinsic facets of social/cognitive interaction/functioning; hence, the methods used to collect data in this enquiry will be trying to procure what happens when the internal interact/s with an external influence/s (Yin, 2006). This illustration provides a start for thinking about yet other types of mixed method research. The point is, if a relationship is completely absent— particularly where two or more methods address wholly different dependent, independent, or descriptive variables—the mixed methods are likely to form separate studies, not a single study (Yin, 2006). All these influences are important and relevant, but they are only some of the processes that, together, comprise a complex social world and unfortunately; understanding that the relevance and value assigned to learning by adults’ highlights the importance induced, does not necessarily liberate them (Hacking 1999; p. 2) from any disenfranchisement they could feel. Likewise, the researcher understands that the aforementioned factors are not the only variables that are existent; however, the researcher is of the opinion that those factors (see fig. 3 + 4) are the most prominent from the observations made and literature review conducted. Research Design Fig. 2 is illustrating the design and flow of data analysis that establishes the internal validity, reliability and quality of the research enquiry. Historical background Considering participation in adult learning since 1996 we see it has remained around 40% for those of working age (16 – 69) for seventeen years. These were either currently participating, or had recently participated in the last three years. Of those that did participate, there is an equivalent amount that has not participated since leaving full time education. Although, 80% of students’ currently participating intend on continuing in further education after they have completed the present course (see Tab.1). Whilst participating in Further Education and Lifelong Learning I observed a possible explanation for the existence of these variances (that being relevance and value). A possible explanation for the disordinal interaction (percentages decrease in the ‘Likely to learn in the future’ group whilst percentages for ‘Unlikely to learn in the future’ group increase) demonstrated in table 1 could be; the further in years an adult moves away from education the less relevance and value they attribute to returning to it. Or, is it as Siraj-Blatchford (2010) may suggest; that the adults are overscheduled and more committed to sustaining the home environment and maintaining a career with ‘on the job’ training. Multimodal Heuristics Informal learning is seemingly multimodal i.e. being valuable and relevant to the matter at hand and socially constructed through long/short term interactions (GTC 2006). The informal learning mechanisms that mediate influence shapes learning environments’ (Evans, et al., 2010; p. 6), cognitive processes and our social interactions (Evans, et al., 2010; p. 6). ‘Meaning’ then, is co/re-constructed by experience resulting in the multiple interpretations that create the social reality in which people act (Flowers 2009; p. 3). And as Vygotsky would state; context affects cognitive and by way of behavioural activities (De Abreu, 2000; p. 3) Bruner’s suppositional framework suggests that learners form new ideas or theories based upon what they already know (GTC 2006). His theory of learning, not only, related to the way childrens thinking developed, but it could also be applied to adults learning new and unfamiliar material (GTC 2006). Learners, as Bruner proposes, are creators and thinkers through the use of inquiry (GTC 2006). The process of which how learners dynamically construct knowledge is heavily in focus: implying the transformation of information, which suggests that Bruner’s theory of Constructivism falls into a cognitive domain (GTC 2006). Learners are provided with opportunities to construct new knowledge and new meaning from authentic experiences (Brockmann 2011). As a result, this exposes the pivotal role Multimodal Heuristics start to have when adults’ decide to return to education. For instance, a parent can reassure a frightened child that ‘shadow monsters do not exist!’ Although, a sibling can suggest leaving the light on to scare the monsters’ away. This indicates that informal learning can alter our worldview (e.g. ‘When did you stop believing in Santa?’) if it is seen to offer a plausible solution. This supports the concept of how informal learning can contribute to our understanding, cognitive processes (De Abreu 2000), social interactions, and the associated behaviours (Schwartz 1995; p. 5). These multimodal components; not only determine the level of commitment and motivation (Park Choi 2009) that is ascribed to the retention of relevant and valuable information (Gibbons Bylsma, 1984; p. 23), but also contributes to the ease of transfer and retrieval of that information (Ekey 2012). The characteristically pragmatic nature of adult learners’ (Abdullah, et al., 2008; Kohl-Frey and Schmid-Ruhe 2007; Crittenton Women’s Union 2012) also demonstrates this need/requirement for information to have applicability to their life. This is determined by the perceived applicability it has to their future experiences and interaction. The internal dimensions of meaning-making are also multimodal (Clark 2011) and seemingly derived from the combination of the value and relevance (or Multimodal Heuristics adults’ decide, through cognitive appraisal, their own level of involvement) assigned by the adult to measure applicability. Consequently, we could suggest that this is an ad hoc contribution to our social cognition (Aronson et al., 2005; p.57 – 64; De Abreu 2000; p. 4), our availability heuristics (Rules of thumb; Aronson et al., 2005; p. 74 75) and the associated behavior and schemas (Aronson et al., 2005; p. 59 61), which then assist navigation of social environments’. Unfortunately, understanding that the relevance and value assigned to learning by adults’ highlights the importance induced, does not necessarily liberate adults’ (Hacking 1999; p. 2) from the disenfranchisement they could feel in institutions where learning is delivered primarily from a traditionally pedagogical approach. Similarly, these interactions are situational and experienced directly by participation, so it will be difficult to generalize the results further than adult learning. Theory development Essentially, humans tend to seek out information that confirms what they think/believe to be most relevant or true to their experiences and/or future interactions; a relative cost-benefit/means-end (Evans, et al., 2010; p. 6) cognitive appraisal that enables Multimodal Heuristic co/re-construction (Clark 2011). This process begins to filter out information that is considered worthless. The cost-benefit (Primary appraisal) and means-end analyses (Secondary appraisal), along with the personal value and relevance adults’ assign to learning (‘rule of thumb’ Gustafsson, L., Mouwitz, L. (2008); p. 5) appear to be hierarchical and Maslowian in nature. Additionally, an adult must consider, through means-end analysis, the benefit of actively participating and building upon their knowledge and experience, throughout their participation in learning. Ultimately mediating their need for satisfaction i.e. Socio-emotional negotiation and selectivity (Houde 2006). As a result, for the adult to consider participation Multimodal Heuristics must negotiate support for expectation and assess the benefit knowledge, learning and education have in recompense for reorganizing multiple obligations, and competing priorities (Evans, et al., 2010; p. 12). Therefore, is socio-emotional negotiation and selectivity a process of fragmenting information so that it creates a heuristic commensurability with an individual’s normative social and cognitive functioning, which therefore, influences behaviour i.e. influential connectivity of socio-cognitive interactivity affecting the potentials for action? Fig. 3 is showing the internal framework of the decision making, and meaning-making, mechanisms that help generate mental constructs of multimodal heuristics. To some degree, we can compare the assessment of value and relevance to Gustafsson Mouwitz (2008) description of Proficiency Theory, and, means-end and cost-benefit analyses to McClusky’s Margin Theory (1974, as quoted in Gibbons Bylsma 1984). These theories emphasize a need to be competent at tasks’ whilst being realistic about certain physical, mental and social capabilities. If there is conflict between primary and secondary appraisals this could be seen as a violation of expectation (Deffenbacher 1993), which may account for drop-out rates, serial signers’, absenteeism, non-participation in task relevant activities, specific course popularity, the cost-benefit/means-end analysis (Evans, et al., 2010; p. 6; Geertz 1993; p. 4 5) for staying the course and societal perception of lifelong learning (Tab. 1). For instance, after asking my students’ (12 in total) if they had any questions about what had been learnt, they responded with â€Å"what would I do if†¦?† and â€Å"When would I use†¦?† As there were only subtle variations in discourse, in regards to relevance and value, I feel this highlights (1) what comprises Multimodal Heuristic co-construction, and (2) what is required from information when it is presented outside of their interpretation of it. Moreover, adults maintain autonomy (Gibbons Bylsma 1984) by performing a cost-benefit analysis to justify their participation; being that peripheral or full (Swan 2005; p. 5). Firstly this, amongst others mentioned, will form the basis of ‘what counts as value and relevance evidence’, and, from which, quantitative data will be collected (questionnaire). Lastly, the quantitative data will be qualitatively complemented with a semi-structured interview to produce a rich narrative and attain thick descriptions (Geertz 1993). The semi-structured interview will be conducted with a subset of the surveyed group and will represent a cross-section of the adult learners’ in that group i.e. single parent, co-parent and a single male/female with no dependants. And as Denscombe (1999) and Brockmann (2011) found; interaction is situational and experienced directly by participation, making it essential to respect [their] views, with, further recognition given to the possibility that [their] priorities may not reflect the general consensus view or official theory. For example, Gustafsson Mouwitz (2008) have reported; what is valued and encouraged in formal learning environments lacks to varying degrees explicit relevance in the workplace. Therefore, adults must demand a greater degree of relevance, value and satisfaction when deciding to return to, and participating in, education (Abdullah, et. al. 2008; Houde 2006). Fig. 4 is showing the internal framework of secondary appraisal that aims to justify the decision made and validate the perception of learning by paralleling meaning-made with the realities of the study. Illustrating, not only that the individual agency of these interpretations of relevance and value are co/re-constructed (Clark 2011) cognitively (GTC 2006) and socially (Hacking 1999), but also that adults’ apply this form of Heuristic Multimodality when seeking satisfaction from having their expectations fulfilled. Park Choi (2009) have reported that relevance and satisfaction, being sub-dimensions of motivation, are known to be interrelated with various course-related issues. Even though the societal influences mentioned in this report can modify (1) the assessment of relevance and (2) affect the personal satisfaction adults cultivate (Park Choi, 2009) they can also mediate and reinforce participatory behavior (Park Choi, 2009) by enhancing the importance adults’ induce when deciding an academic and/or social level of involvement (Gibbons Bylsma 1984). Furthermore, students’ have asserted that relevance is a significant mediator in their assignment of value. Many students’ have commented that relevance paralleled the value assigned to learning and their specific choice of subject(s). These statements were observed over time and place using a relative constant comparison method (Cohen et al., 2000; p. 151). Their comments demonstrated the application of a cost-benefit and means-end analysis e.g. â€Å"How relevant is†¦in the big scheme of things?†, â€Å"When would I use†¦?† and â€Å"I don’t see the relevance? Evidently, the use of Multimodal Heuristics acts as a mechanism that could also increase commitment, dedication and motivation (Park Choi 2009). In constant comparison data are compared across a range of situations, times, groups of people, and through a range of methods (Cohen et al., 2000; p. 151 2). The process resonates with the methodological notion of triangulation. The constant comparison method involves four stages: Comparing incidents and data that are applicable to each category, comparing them with previous incidents in the same category and with other data that are in the same category Integrating these categories and their properties Bounding the theory Setting out the theory The subjective ontological/epistemological view, research design and methodology exhibited in this report is sufficient and necessary to explore this direction of enquiry, if it were absent, it would prove problematic supporting a theory with an accompanying objective approach that advocates detachment (Flowers 2009; E891 Part 2: Action 2.2; Gage 1989; E891 Part 2: Action 2.5), when, in this case, it is more advantageous to explore the subjectivity of individual agency, participatory behaviour and situational experiences, motivation, and, the personal value and relevance assigned to learning, as these are closer to the truth. Instrument Design There will be two distinct phases to data generation; firstly, questions will be formulated from each of the four themes that CAL, Andragogy and Margin and Proficiency theories appear to create and then randomly assigned (Nederhof, 1985) to a questionnaire. A descriptive analysis of each question will be conducted to address whether the aforementioned multi-dimensionalities of adult learners’ are being considered. The strength of the trend in the agreement/disagreement should build a picture of the shared experiences. These questions will then be relocated back to the themes that created them, scored (Likert Scale; the higher the score the more relevance and value is attributed) and compared with the descriptive analysis to, not only generate a semi-structured small group interview schedule (Gillham, 2000), but also to get a sense of what is personally valuable and relevant about learning. This is an attempt to demonstrate; how meeting these multi-dimensionalities may be instrumental in maintaining learner participation (Park and Choi 2009). Furthermore, by mapping these realities, establish whether they support the general consensus view of these adult learning theories. An opportunity sampled group (16 – 35+) will be surveyed using this questionnaire (13 in total) with a small group interview being administered to a subset of the surveyed group (5 in total). Ideally, this subset should be representative of the adult learners’ in that educational facility. Even though the whole group will be opportunistically surveyed; in phase 2 every effort will be made to be more purposive. In order to support internal validity and ensure the reduction of any bias the incorporation of a ‘social desirability’ measure (Nederhof, 1985; SDR) will be added to the questionnaire. Certain questions will be cross referenced with one another to assess whether the adult learners’ are responding in a socially desirable way. This local blocking technique should increase the internal validity of the questionnaire, enhance the internal consistency of the small group interview questions, reduce bias and maintain rigour when all the data is analysed. This should also allow individual agency (E891 Part 2: Action 2.4; Gage 1989; Denscombe 1999), shared experience and the personal value and relevance attributed to learning to be highlighted. Due to the amount of data that could have been reported the evaluation will be specifically limited to the triangulation narratives of the ‘Theme Summaries’, interview data i.e. content and descriptive analysis (Clark, 2011). The researcher firstly formulated questions from these naturally occurring themes and searched for consistencies and inconsistencies (Denscombe, 1999) between the summary narratives (Gillham, 2000) and statistical outputs from the descriptive analyses (Bryman, 2007). Phase 1 As there were 30 questions generated from the four themes the in-depth analysis of each question will be triangulated and presented in the theme summaries. In an attempt to expose any consistencies/inconsistencies (Denscombe, 1999; p. 217-8) in the responses the data will be compared against the learning theories that created them: ensuring validity. Consequently, due to the amount of quantitative data generated from the in-depth analysis of the individual questions, this report will only include the second stage of Phase 1 i.e. descriptive analysis and theme summary triangulation. The interview responses from Phase 2 will be further triangulated with these summaries and content analysed to highlight the adult learners’ realities and ascertain what influences their decisions and motivates them to return too education i.e. by constant comparison method. Theme Summaries Social contact and Relationships Q1, Q2, Q6, Q7, Q17 Q19 and Q30 The adult learners’ appear to value social interaction and feelings of reciprocal respect whilst participating in learning, which demonstrates that the adult learners’ value a sense of ‘belonging’ (16/21). However, there is a small percentage that does not see ‘belonging’ as being of value. Therefore, the feelings of reciprocal respect and support cannot be generalised as influencing their decision to continue in learning. Internal expectations Q10, Q13, Q14, Q15, Q18, Q22, Q26, Q28 and Q29 This theme relates to the adult learners satisfaction. Satisfaction, being a sub-dimension of motivation, is something that must be regarded as paramount in the adult learning experience. The consistent attendance of the adult learners’ at the session/s is testament to their satisfaction with the course and the delivery thereof (18/27). In essence, if the adult learner considers that the potential learning opportunity is not transferable to the workplace, is not satisfied, or perceives it as inadequate at providing improvement to their problem solving capabilities could ultimately diminish their motivation to participate. Goal and relevancy orientation Q3, Q4, Q5, Q9, Q11, Q16, Q20, Q23, Q25 and Q27 External expectations Q8, Q12, Q21 and Q24 As these last two dimensions, respectively and comparatively, share a greater degree of similarity they will be interpretatively combined and presented together. Looking at these from a political perspective; the demand for lifelong learning to have greater prevalence in society sets an industry standard that demands conformity to it. Subsequently, this appears to facilitate the re/co-construction of self-directedness and the personal interests of adult learners’ so that they begin to mirror ‘what is required of them’; which is indicative of a cost-benefit/mean-ends analysis. Therefore, some adult learners’ might be so focussed or motivated on getting the qualification that they adjust their sense of self-direction in order to reorganise their lives and satisfy what is required of them i.e. Mutability for the betterment of self. It could also be suggested that the pressure too have certain qualifications encourages participatory behaviour in some adult learners’ and determines the relative conformity to industry demands and learning the required skills i.e. something they adapt to rather than adapted for them (Q8, Q9 and Q10). Conformity, in this sense, would then act as a pre-determinant to achievement; the perception of economic sustainability and upward mobility and what value, and relevance, learning has. Not surprisingly, this could be one reason why thousands of people leave their jobs: they only took the job because it is what was demanded of them, which is in direct conflict with their personal interests, self-directedness and life goals. Which also illustrates that cognition can be influenced by social interaction and be co/re-constructed by experience and meaning-made. The questionnaire included items that let the participant assess the value and relevance they attribute to learning as an adult. The overall strength of this agreement was guided by their experiences as an adult learner. However, some of the diagnostic questions seemed to be complex and ask two things of the participant. As this is a major source of error (Hammersley et al., 2003) the validity of those questions will be scrutinised as the participants may have weighted one aspects of the complex question more important than the other aspect, hence, an adumbrated response i.e. a decrease in validity. However, all of the responses were reduced to one mean average for that individual question, and as these were pooled from the four themes that characterise adult learners’ it reduced sampling error and bias. Furthermore, as there was a two stage analysis in phase one the validity of the research instrument is strengthened; especially when we factor in the use of the SDR measure to control for bias (Nederhof, 1985) and the encouragement of omission (Hammersley et al., 2003) when the participant had no opinion. What we cannot suggest at this stage of the analysis, however, is that the shared experience led to a shared meaning. As Denscombe (1999) ascertained; the perceptions of the individual are not always consistent with the general consensus view (Brockmann, 2011) of the group as a whole. This extends to the meaning-made and the individual nature of the meaning-making process. The surveyed group cultivated differing levels of relevance and value from their shared experiences. But, this was seemingly determined by the level of relevance and value that was extrapolated from their continued participation. Hence, the individual agency of meaning-making is an outcome of the level of value and relevance attributed to the shared experiences of learning as an adult, the level of satisfaction and a sense of mutability for the betterment of self. Phase 2: Narrative of Qualitative data Before the triangulation, constant comparison and content analysis the researcher must point out that (1) this was a small scale study, (2) the interviews was held in a small group so full disclosure by each participant was not always possible and (3) the results should not be over generalised to other adult learning situations. All that is being sought is an insight in to the connectivity of socio-cognitive interactivity and the subsequent influence on representative individuality; the multidimensionality of participatory behaviour and what the adults felt their motivations for returning to education were and still are. This should (1) map the external/internal influences on the adult learner; (2) expose the dimensions behind this seeming connectivity of socio-cognitive interactivity that create the potential/s for designated types of action i.e. Multimodal Heuristics and (3) if the analysis supports the researcher’s theory and the adult learning theories that feature in this enquiry. Some of the interviewee’s shared a meaning to one degree, but had a different meaning-making process before reaching that decision; the salient feature was a shared-meaning in a shared-goal in reaching university or attending a higher level course from the successful completion of the current course of study. This gave them a common ground on which to build upon ‘what learning means’ to them on an individual basis whilst allowing the shared-meaning element distinguish and define their individual social relationships in the class; whom they sought clarification from; what level of involvement they chose and what comparative judgements they begin to make on others in the session/s. Walter: â€Å"Well I think if you do†¦ I think if you do†¦like, we are social people, things†¦we are social and that’s that, that’s what we are†¦we are designed to be social people, if we exclude ourselves we do not, you know, we lose all basic human function, it’s like the guy at the front, you know he doesn’t †¦ he can exclude himself, he doesn’t do anything, he doesn’t enjoy being here, doesn’t have any excitement about coming and learning†¦if you exclude yourself from everyone else you’ll probably not learn!† Serena: â€Å"I like learning with a group but then it’s dependent on what I do with that information†¦but when it’s writing things down or posters and stuff I can’t have other people touching.† This illustrates that the need to feel self-directed and sometimes being free from outside interference is just one of the commonalities we start to see in the participants responses. Although, some of the interviewee’s do highlight that; Sally: â€Å"I came because I needed to do it, but now I quite, I’ve more motivation to do it because I enjoy it.† Therefore, the individual agency of meaning-making is, not only an outcome of the level of value and relevance attributed to the shared experiences of learning as an adult, the level of satisfaction and a sense of mutability for the betterment of self, but also the means/ends-cost/benefit interconnectivity seen in their decision-making process i.e. motivated to do it and their continued participation in the session/s that seemingly contributes further to the mutability for the betterment of self in these adult learners. The individual meaning-made is a product of these connective interactivities varying – and individually decided high/low levels of cost/benefit the course has and what perceived means/end reward the course provides for successful completion i.e. the multimodal heuristic factors that led to participatory behaviour and satisfaction. The shared-meaning is an accidental affinity that becomes synchronous with other people that are pursuing a similar goal as them. This suggests that they share similar educational values and relevancies due to their common or shared goals. The shared-meaning dimensions presumably start to mediate the differing high/low connective interactivity level in the cost/benefit and means/end analyses. Furthermore, this also starts to define and distinguish individual social relationships; who we seek advice and clarification from; what level of involvement we chose and what comparative judgements we make on others i.e. asynchronous affinity with others. The comments from these interviewee’s also highlights the need to feel proficient and competent about the material in the course and where they culture this confidence. It seems the more confident the adult learner gets about understanding the material and being able to discuss, question and seek clarification on their understanding the more proficient and competent they feel. This bolsters their feelings of satisfaction and adds to their motivation to continue through reducing the physical and mental sense of effort i.e. cost and/or means and increasing the perception of benefit cultured from continuation in the session/s. e.g. internal expectations and social relationships. This could be defined as a beneficial compromise for the betterment of self being the mediation of the perceived value social contact offers in raising confidence, increasing feelings of proficiency and reducing our fear of incompetency. The commentaries also point toward Margin theory (Gibbons Bylsma, 1984) in the manner of how ‘power’ and ‘load’ i.e. the amount we can manage is balanced with the effort we can assign to it and ‘expansive’ and ‘restrictive’ perceptions of future time i.e. the older you are the more urgent something becomes also contributes to the motivations of the adult learner (Gibbons Bylsma, 1984). So, is motivation the product of a restrictive ‘future time’ perspective creating a behaviourally urgent response to the realisation of your current educational inequities; therefore, adding to the perception of the reduction in opportunities for sustaining economic upward mobility? For example the following conversation illustrates the reasoning behind this question; Researcher: ‘so does anyone find, you know, that helps them decide to do a course, or, was it a combination of both things were like valuable and relevant to you as well?’ Walter: ‘Yeah, yeah†¦that’s the reason I’m here, you know you can’t get a well-paid job without English and Maths!’ Leroy: †¦Ã¢â‚¬â„¢and without those I can’t precede on to university’ Researcher: ‘So you can see the as an industry standard kind of then?’ Walter: ‘Yeah, this is the industry standard’ Researcher: ‘So to actually progress you need these things to progress?’ Walter: ‘Yeah†¦yeah†¦Ã¢â‚¬â„¢ Leroy: ‘Like to myself, like to have this qualification would make me feel better about it†¦but,’ Researcher: ‘Yeah†¦Ã¢â‚¬â„¢ Leroy: ‘†¦it’s a requirement’ Researcher: ‘yeah like a stepping stone’ Leroy: ‘yeah.’ Therefore, these adult learners’ may just see the benefit of having the qualification to progress beyond where they are now. This could also suggest that these adults’ are fully aware that the ‘real world’ applicability of certain subjects are determined by the industry demand for that subject, making a qualification economically more relevant and valuable to these adult learners’. We could theorise that society has a shared understanding about what industry requires of the workforce and how this requirement places a demand on the learner to rearrange their lives in order to participate in learning. Therefore, shared meaning in society could be facilitated by a shared understanding of what it demands of society, which supports the theory that adult learners’ must assign more personal relevance, value and expectations of satisfaction to learning before there is the motivation to return to education i.e. is there a beneficial compromise between what I want and what they require. And as can be seen in the descriptive analysis of Q20, Q21 and Q22.These questions relate to social influence and societies perception of value and relevance assigned to learning. The adult learner agrees that the decision to attend a course of study was suggested to them (Q20) and that this social influence/encouragement essentially provides the persuasive reinforcement to their implicit understanding that; learning increases an adult’s chances of employment (Q21). The adult learners’ also feel that the support they receive from the different sources of this social influence/encouragement is at a level which permits their participation on the course of study. We could again theorise that an individual knows what is demanded of them in the employment market, but they seek confirmation on what they already know. This suggests that ‘meaning’ is socially co/re-constructed by the individual seeking confirmation on their present understanding in order to reinforce their decision, and by way of, increase motivation to return to learning. Furthermore, the adult learners’ do not feel they have to make allowances to attend a course, as long as the scheduled session/s is at a convenient time for them to attend i.e. the conscious effort to avoid the conflicts between personal obligations and scheduled session/s. Moreover, showing that, for these adult learners’, the course of study has value and is personally and economically relevant to them. And as it was outlined in the ‘Theory Development’ section of this report; we can compare the assessment of value and relevance to Gustafsson Mouwitz (2008) description of Proficiency Theory, and, means-end and cost-benefit analyses to McClusky’s Margin Theory (1974, as quoted in Gibbons Bylsma 1984). These theories emphasize a need to be competent at tasks’ whilst being realistic about certain physical, mental and social capabilities. Moreover, because the theories that were used in this study have overlapping dimensions (e.g. Q3, Q4 and Q12 overlap Social contact and Relationships; Goal and Relevancy orientation and External Expectations) with each dimension seemingly providing a piece to the decisional mà ©lange that affects the internal expectations i.e. individual agency of the adult learner. We could therefore suggest that Multimodal Heuristics and co/re-constructive social influence, not only becomes more evident when motivation towards participation is being established and/or maintained, but may also be one of the key components in the processes that assist the transformation of identity. Hence, as a sense of belonging, competency, proficiency and satisfaction are valued and relevant to the adult learner and evolve as they evolve; as do their identities. Equally, in a sociocultural ontology progress in learning is viewed along trajectories of participation and growth of identity, so both competency and belonging matter in understanding learning. It is for these reasons that a sociocultural ontology describes learning as a transformation of identity. And as the report is looking at the macro and micro-structural influences on the adult learner and how that comes to mediate and motivate them toward participation we can suggest quite firmly that identity transformation is closely tied to multimodal heuristics which is apparently mediated by a co/re-construction between the connectivity of social/cognitive interactivity thus having an impact on the identity formation of the adult learner. So the relevancy and value that is selected from external sources is fragmentally factored from differing micro and macro-structural influences and negotiated in to cognitive constructs i.e. internally mediated hence facilitating the decisional components that create the motivation for, and support continued participation towards, designated types of action. Discussion, implications and conclusions So, are social contact and relationships the result of synchronous affinities? Is the use of multimodal heuristics an actual contributor to identity transformation? All that can be suggested is that the results support the theory of multimodal heuristics and the connectivity of interactivity and imply that motivation is established through the individual deliberately, and sometimes vicariously, extracting information from these proximal and distal influences. Hence, social/cognitive collocation comes to, not only reinforce their decision to participate in designated types of action, but also – to a greater or lesser extent impacts upon the transformation of identity. But, we must keep in mind that this is paralleled with a balance between the beneficial compromises for the betterment of self and the perceived value social contact i.e. belonging offers in raising confidence, increasing feelings of proficiency and reducing the fear of incompetency. The real implication of these results is the noticeable benefit of informal conversations being used to reinforce learnt knowledge. The participants suggest that more time for reflection and confirmation would go some way to aiding the retention of new information, how the information actually relates to their personal circumstances and how this also contributes to feelings of belonging, proficiency, competency and feelings of increased confidence. For example; Walter : â€Å"†¦if you have a conversation with someone, say after this class, you’ll remember that conversation better than you would, you know than someone standing at the front of the class going ‘this guy wrote this poem about this† And like the small child that is afraid of ‘shadow monsters’ and leaves the light on; the sessions could benefit from the incorporation of small group/whole class learning reinforcement dyads of informal conversations. This would then start to determine the level of commitment and motivation (Park Choi 2009) that is ascribed to the retention of relevant and valuable information (Gibbons Bylsma, 1984; p. 23), and further contribute to the ease of transfer/retrieval of the current learning material/s and any new information (Ekey 2012). In conclusion, if adults are autonomous, self-directed and pursue their personal interests and goals then; when an adult decides to return to education the course of study must display a greater degree of relevance to the adults. If the course of study is perceived as having relevance, it (1) fulfils their need for feeling autonomous, (2) allows the adult to make an informed decision as to the value it has, (3) contributes to the continuation of feelings of self-directedness and (4) also contributes to their perceptions of being closer to achieving their goals’; thus adding value. Especially when we factor in that adult learners’ are complying with requirements’ laid down by someone else and may need to reorganise multiple obligations and competing priorities in order to participate. Another reason that these adult learners’ generally value the social interaction, support and reciprocal respect they receive whilst attending a course of study. Hence, the sense of belonging would be enhanced if there were more opportunities for interaction. Therefore, creating more opportunities for reflection between learners’ could, not only, reinforce learning, but also support the feelings of belonging through increasing the opportunities for discussion on how the material covered in that session/s contextually relates to them. Moreover, focussing on enhancing feelings of proficiency by allowing the adult learner to co/re-construct their current understanding through reflecting upon it with learners’ that share the same learning experience and synchronous and asynchronous affinities. References Abdullah, M, Parasuraman, B, Muniapan, B, Koren, S Jones, ML. (2008) ‘Motivating factors associated with adult participation in distance learning program’ International Education Studies, 1 (4), pp. 104-109. [online] http://ro.uow.edu.au/cgi/viewcontent.cgi?article=1569context=commpaperssei-redir=1referer=http%3A%2 ‘Mutual Research Designs: Redefining Mixed Methods Research Design’, pp. 29 – 35, Taken from ERCM (2007) ‘6th European Conference on Research Methodology for Business and Management Studies’ Universidade Nova de Lisboa, Lisbon [online] http://books.google.co.uk/books?hl=enlr=id=VtYcSTUV0nQCoi=fndpg=PA29dq=bryman+pragmatism+%22multi+methods%22ots=bC6CHJujM9sig=sWf3Hfxlkrz-79fe5K0eGeqLI3M#v=onepageqf=false (accessed on 18/06/2013) Aronson, E., Wilson. T. D. and Akert, R. M. (2005) ‘Social Psychology’, 5th Edition, New Jersey, Pearson Education Inc, Cpt. 3, pp. 59 – 61, Cpt. 3 pp. 57 – 64, Cpt. 3, pp. 74 75 Brannen, J. (2005) ‘Mixed methods research: a discussion paper’, ESRC National Centre for Research Methods, Review Paper 5. [online] http://eprints.ncrm.ac.uk/89/1/MethodsReviewPaperNCRM-005.pdf (accessed on 01/08/2013) Brockmann, M. (2011) ‘Problematizing short-term participant observation and multi-method ethnographic studies’, Ethnography and Education, vol. 6, no. 2, pp. 229–43. [online] http://www.tandfonline.com.libezproxy.open.ac.uk/doi/pdf/10.1080/17457823.2011.587361 (accessed 18/04/2013) taken from The Open University (2013) ‘E891 Action 3.10: Short term Ethnography; Part 3: Research design and data production, Milton Keynes, The Open University Bryman, A. (2006) ‘integrating quantitative and qualitative research: how it is done?’ Sage Publications, London, vol. 6(1) 97–113. [online]

Isolation of Recombinant Escherichia Essay Example for Free

Isolation of Recombinant Escherichia Essay One technique important in both genetics and biochemistry is the Polymerase Chain Reaction (PCR), first developed in the 1960s, and then automated in 1983. Current PCR technology was not developed until the discovery of thermostable polymerases, specifically Thermus aquaticus (Taq) polymerase (1). The protein Taq polymerase was first isolated from the extreme thermophile T. aqauticus, where extreme thermopiles are bacteria that live in temperatures at or above 45 °C. The Taq enzyme is a member of the DNA polymerase I family (2, 3). The interesting property of Taq polymerase is that it has a temperature optimum at 74-75 °C, allowing it the remain active in temperatures required for PCR double stranded DNA denaturation (3, 1) . The protein has an approximate molecular weight of 6263 kDa when isolated from T. aquaticus, and 94 kDa when isolated from recombinant Escherichia coli, and is still stable at temperatures of 93-95 °C, hence the thermostability of the enzyme ). Taq specifically lacks any proofreading activity in the 3’ to 5’ direction, and therefore has a relatively high error rate of single base mispairings of 1 error per Isolation of Recombinant Taq Polymerase for PCR 9000 nucleotides, as well as a frame shift error rate of 1 per 41,000 basepairs (5, 6). Taq polymerase has an activity that is highly dependent on the environment of which it is in as it is thermostable, and has differing activities at nearly all temperatures up to the point of denaturation. Taq specifically can add up to 1000 base pairs in length on a template in under one minute under typical PCR conditions. The enzyme has a specific activity of 200,000 units mg-1, and can add approximately form 60 nucleotides per second at 70 °C (7). The isolation of Taq is essential for the PCR reaction. The most important reason for Taq being used in PCR is the thermostability at high temperatures (95 °C). This allowed for the process of elongation, annealing, and denaturation to occur without the replacement of new enzyme, and thereby, was more efficient, faster, and cheaper because the reaction could be automated through the use of a machine known as a thermocycler which basically is just a machine able to change temperatures of an isolated environment rapidly (7). Prior to the discovery of Taq, PCR was done using Klenow fragments of E. coli DNA polymerase I at 37 °C. The lack of thermostability required replenishment of enzyme after each PCR cycle (8). One of the initial difficulties of Taq polymerase was the organism in which it was expressed in, T. aquaticus, as it was difficult to culture and produce large quantities of enzyme. E. coli bacteria were engineered to expressed the Taq polymerase gene to allow for retrieval of large quantities of enzyme ). The isolation of the Taq gene involved culturing T. aquaticus and then isolated the DNA of the cells through lysing, proteinase K addition, extracting of aqueous and phenolic phases, dialyzing of extractions, addition of SDS, and then centrifugation of solution to eventually retreieve the DNA of the organism as outlined in Lawyer et al., 1989. With the isolation of the 2401+ BP gene of Taq, the gene was incorporated into a 6.58 kbp plasmid (pLSG1). The gene was inserted 171 bp distal to the lacZÃŽ ± promoter/operator, and 109 bp distal to the BgII site, so the gene expression could be controlled through an inducible promoter. With the pLSG1 plasmid, the vector was introduced to E. coli bacteria to allow for plasmid uptake (4). Other experiments have been conducted towards the purification of Taq from recombinant E. coli. Specifically Engelke et al., 1990 developed a method for purfication of Taq. The E.coli strain 2 DH1 was used for the expression of the recombinant plasmid containing Taq polymerase. The bacteria were grown in 12 Litre batches of Luria Broth; using 1 mL of saturated DH1 culture and 80ÃŽ ¼g/mL of ampicillin. Isopropyl-1-thio-ÃŽ ²-Dgalactopyranoside (IPTG) was added to 0.5mM and the cultures were grown for 16-20 hours. The cells were harvested in 2.4 L of buffer A (50 mM TrisHCL, pH 7.9, 50 mM dextrose, 1mM EDTA) and collected via centrifugation, resuspended in Buffer A with 4mg/mL lysozyme and incubated at room temperature for 15 minutes. Buffer B (10 mM TrisHCl, pH 7.9, 50mM KCl, 1mM EDTA, 1mM phenylmethylsulfonyl fluoride (PMSF), 0.5% Tween 20, 0.5% NP-40) was added and incubated in 180 mL fractions, for 60 minutes at 75 °C in a water bath. The mixtures were centrifuged at 8000 rpm for 15 minutes at 4 °C. Taq then precipitated with polyethyleneimine (PEI) at room temperature, then isolated through centrifugation and suspended in buffer C (20mM HEPES, pH 7.9, 1 mM EDTA, 0.5mM PMSF, 0.5% Tween 20, 0.5% NP-40) containing 0.25 M KCL. PEI eluatents were diluted in 50mM KCL and buffer C and applied to a 150mL BioRex 70 ion exchanger column, and then eluated using 200mM KCL. The protein was dialyzed for 12 hours against two changes of 1 L storage buffer (20mM HEPES, pH 7.9, 100 mM KCL, 0.1 mM EDTA, 0.5 mM PMSF, 1mM dithiothreitol, 50% glycerol. The experiment resulted in 40-50 mg of protein per litre of cell culture (9). The methods used in this experiment differed in certain key aspects. First, Engelke’s experiment made use of a higher concentration of ampicillin. The IPTG was added to the same concentration, but was added after cell growth up to an optical density of 0.700. Instead of a water bath at 75 °C, this experiment made use of an air incubator for the temperature requirements. Engelke’s experiment made use of PEI to precipitate Taq, while this experiment made use of 30g of (NH4)2SO4 per 100mL of supernatant. Buffer C was not used throughout this experiment, and no ion exchange columns were used. The dialysis procedure was done for twice as long with twice as many changes of solution per 6 hours. The changes made from Engelke’s experiment offers a different method for protein precipitation. The method used by Engelke made use of PEI which is an affinity precipitation method versus a salt prec ipitation method. The PEI Isolation of Recombinant Taq Polymerase for PCR method has the major drawback through the lack of selectivity, and can often precipitate nucleic acids as well (10). This is why the BioRex column needed to be used. Ammonium sulfate has the advantage that the precipitation can be controlled based on ionic strength of species involved, as well as has no negative effects on the activity of the target enzyme. Salting out also has the advantage that only native state proteins are precipitated due to the hydrophobicity involved with native state proteins (10). Buffer C was not required for this experiment as no BioRex column was required. This experiment made use of various techniques and methods including: SDS-PAGE, differential centrifugation, Western Blotting, real time-PCR (rtPCR), PCR, agarose gel electrophoresis, and dialysis. Two important techniques were PCR and rt-PCR. PCR does not allow for the quantification of DNA amplicons as it is an end-point PCR, but it does allow for confi rmation of template duplication along with measurement of base pair length. Amplification of primer would confirm the presence of a thermostable DNA polymerase. The following agarose electrophoresis helps to find amplicon size which can tell us the activity of Taq, as well as the specificity, as one template should only return one band in PCR (7). rt-PCR allows for a quantitative assessment of PCR, and therefore the kinetics of the reaction, as it detects the amount of amplicons produced in the reaction. The point at which the standard curve reaches threshold in cycle number gives information on the activity of Taq, as a more active sample of Taq reaches threshold earlier. Melt curve analysis also provides information regards DNA amplicons in solution (11). The purpose of this experiment was the test the methods for the isolation of PCR grade Taq polymerase from recombinant E. coli using differential centrifugation, salting out, and heat denaturation following lysation of cells to potentially improve isolation of Taq from past methods. The presence of Taq will be confirmed through Western blotting, and rt-PCR and PCR reactions along with purity will be assessed through SDS-PAGE. The activity of Taq will be found through rt-PCR and PCR. Finding the most efficient method for the isolation of Taq offers a valuable reagent source for any PCR reactions required. The isolation technique would also be applicable to any thermostable proteins. 3 EXPERIMENTAL PROECDURES Isolation of Taq Polymerase Luria broth (500 mL + 100ÃŽ ¼g/mL ampicillin) was inoculated with 50 ÃŽ ¼L of frozen Taq polymerase expressing E. coli cell stock. Incubation was commenced for 12 hours at 37 °C until the Optical Density had reached 0.700. IPTG (0.5 mM or 0.112g/L culture) was added and the culture was incubated for 12 to 14 hours at 37 °C. The 50mL of cells were then centrifuged (4000 RPM x 15 minutes at room temperature) in an Eppendorf Centrifuge 5810, and 5 mL of buffer A (50 mM Tris-HCl, pH 7.9, 50 mM dextrose, 1mM EDTA) was used to suspend the separated pellet. The solution was then centrifuged again (4000 RPM x 15 minutes at room temperature) in an Eppendorf Centrifuge 5810 and the pellet was once again suspended in Buffer A, with an additional 20 mg of lysozyme added. The reaction was incubated for 15 minutes at room temperature. Following incubation, 5mL of buffer B (10 mM Tris HCl, pH 7.9, 50mM KCl, 0.5% Tween 20, 0.5% NP-40, 1mM PMSF, 1mM EDTA) was added and incubated at 75 °C for 1 hour in a New Brunswick Scientific-Innova 40 incubator shaker series, and shaken by hand approximately every 5 minutes. The solution was then centrifuged (15000 RPM x 10 minutes at 4 °C) in a Thermoscientific Sorvall RC 6+ centrifuge and using a 603s Delta Range 30g of (NH4)2SO4 per 100mL of supernatant (8 mL of supernatant equivalent to 2.4g (NH4)2SO4 ) was added and incubated for 10 minutes at room temperature and shaken on the Innova 40 incubator. The lysate was then centrifuged again (15000 RPM x 10 minutes at 4 °C) in Thermoscientific Sorvall RC 6+ centrifuge and the resultant pellet was suspended in 2mL of buffer A. The solution was then dialyzed in a Spectra/Por membrane tubing set at 6000-8000 Da molecular weight selection in 1 L of storage Buffer (50 mM Tris HCl, pH 7.9, 50mM KCl, 0.1mM EDTA, 1mM DTT, 0.5 mM PMSF, 50% glycerol) for 24 hours at 4 °C changing the buffer every three hours. The dialysis solution was then diluted in a 1:1 ratio of storage buffer and stored at -70 °C until needed. Protein Concentration Determination A Bovine Serum Albumin Bio-Rad assay standard curve was prepared (0 –0.3 mg/mL) using Isolation of Recombinant Taq Polymerase for PCR a 1mg/mL stock solution and an Asys Expert Plus spectrophotometer set at 620 nm. Bio-Rad assay was run in triplicate using 20ÃŽ ¼L of protein dilution and 150 ÃŽ ¼L of diluted Bio-Rad Dye Concentrate. 10x and 100x dilutions of the sample prepared previously were made and 20ÃŽ ¼L were used with 150ÃŽ ¼L of diluted Bio-Rad Dye concentrate. The solutions were incubated for 10 minutes and absorbances were tabulated. sandwich was then assembled with an additional ice block in the transfer apparatus. The apparatus was run at 180mA overnight in a refrigerator and the membrane was then stored in TBST buffer (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.1% Tween 20) and refrigerated. 24 hours prior to the primary antibody (Anti-Taq monoclonal Antibody (8C1)) addition, the membrane was blocked in 1 gram of Carnation nonfat dry milk (5% w/v) and 20 mL of TBST Buffer. The primary antibody in TBST with SDS-PAGE 5% w/v nonfat dry milk at a 1:800 dilution of A discontinuous polyacrylamide gel was antibody was added to the membrane and shaken prepared using a Mini-PROTEAN Tetra Cell for 1 hour at room temperature. The membrane was module. The casting stand was assembled for 1mm then washed three successive times for 15 minutes gel and filled with National Diagnostics 12% with TBST buffer at room temperature. The Resolving Gel (Protogel 2400 ÃŽ ¼L, Resolving Gel secondary antibody (Peroxidase-conjugated Buffer pH 8.8 1560 ÃŽ ¼L, dH2O 1974 ÃŽ ¼L, 30% w/v AffiniPure Goat Anti-Mouse IgG (H+L)) was then APS 21ÃŽ ¼L, TEMED 6ÃŽ ¼L), casted to 1 cm below top applied in TBST with 5% w/v nonfat dry milk at a of glass plate, and then 4% Stacking Gel (Protogel 1:2000 dilution of antibody and shaken for one hour 390 ÃŽ ¼L, Stacking Gel Buffer pH 6.8 720 ÃŽ ¼L, dH2O at room temperature. The membrane was then 1830 ÃŽ ¼L, 30% w/v APS 6ÃŽ ¼L, TEMED 3ÃŽ ¼L) was washed three successive times for 15 minutes with casted on top with a ten well gel comb. The TBST buffer at room temperature. The edges of the electrode set up was then submersed in 1 x Trismembrane were dried with a Kim and next 1mL of Glycine SDS Page Running Buffer. To each 20 ÃŽ ¼L Renaissance Western Blot Kit NEN Life Sciences of sample, 20 ÃŽ ¼L of Laemmli buffer (0.5 M TrisProducts, Cat. No. NEL 101 luminol reagent with HCl, pH 6.8, 4.4% w/v SDS, 20% v/v glycerol, 2% 1mL of oxidizing reagent were mixed together and v/v 2-mercaptoethanol, 10 mg/mL bromophneol then applied to the membrane. The membrane was blue) was added and boiled for 3 minutes and then then imaged with an exposure time of 40 seconds cooled for 5 minutes on ice. To one well 7ÃŽ ¼L of using AlphaEaseFC software. New England BioLabs Inc. Prestained Protein Marker (7-175 kDa) was added. To the following PCR/agarose gel electrophoresis wells 15 ÃŽ ¼L of standard Taq polymerase was added, A master mix for PCR was prepared (1x PCR along with 20ÃŽ ¼L of six different samples, with the buffer minus Mg, 0.2mM dNTP, 1.5 mM MgCl2, fifth being prepared the previous year with the same 0.5ÃŽ ¼M forward primer, 0.5ÃŽ ¼m Reverse Primer, 0.1 method of isolation as outlined previously. The gel ng Template DNA and Nuclease-free PCR water) was run at 200 Volts for 40 minutes, incubated in and 22.5 ÃŽ ¼L of master mix and 2.5ÃŽ ¼L of Taq fixing solution overnight and then stained with Bio- sample, the standard, or the Taq prepared a previous Safe Coomassie Blue for one hour at room year were added to PCR tubes and centrifuged temperature under agitation. The gel was then briefly on a Fisher Scientific Accuspin micro 17 analyzed used AlphaEaseFC software. just briefly using 1.5mL eppendorf tubes with no caps to contain the PCR tube. The PCR tube was Western Blotting then added to T3 Biometra Thermocycler and Using the method described above for SDSdenatured at 94 °C for 3 minutes and then 35 cycles PAGE, a SDS-PAGE gel was taken prior to fixing. of PCR with the denature 94 °C for 45 seconds, The gel was then transferred to transfer buffer anneal 55 °C for 30 seconds, and extension at 72 °C (20mM Tris-HCL, pH 8.0, 150 mM Glycine, 20% for 1.5 minutes. The sample was then incubated at Methanol). Immobilon-P transfer membrane with 72 °C for 10 minutes and then temperature was 0.45 ÃŽ ¼m pore size and Whatman paper were cut to maintained at 4 °C. The samples were then stored at the size of the gel. The membrane was wet with -20 °C until agarose gel preparation. A 1% agarose 100% methanol, then transferred to MilliQwater gel w as prepared through 1.5g of agarose (Sigma and soaked for several minutes. A standard blotting No. A-6877 Type II) to 150mL of Tris-Acetate4 Isolation of Recombinant Taq Polymerase for PCR EDTA (TAE) buffer. The solution was microwave for 1 minute and mixed until in solution. Once cooled to 60 °C, 7.5 ÃŽ ¼L of Biotium Gel Red Nucleic acid stain was added and mixed. The solution was then poured into the electrophoresis tray; a comb was installed, and set at room temperature. One Litre of 1x Tae buffer was prepared through dilution of 50x TAE buffer and then the solution was poured onto the electrophoresis tray to cover the gel in 1mm of buffer. 20 ÃŽ ¼L of PCR product prepared previously and 4ÃŽ ¼L of Gel Red dye were mixed and 20ÃŽ ¼L of each sample, the standard, and Taq prepared the previous year and Invitrogen life Technologies 1 Kb DNA ladder Cat. No. 15615016 was run at 150 Volts, 100 mA for one hour (or until dye reached the bottom of the gel). The bands were then visualized under 300 nm light and fluorescence was measured at 590 nm. The gel was analyzed using AlphaEaseFC software. concentration of the sample Taq was 1.88 + 0.11 mg/mL. The solution of proteins was not pure Taq as confirmed by the SDS-PAGE (Fig. 2) as various proteins created distinct bands (B to K excluding E). The standard Taq revealed only one band (A), indicating band E was most likely belong to Taq, as it was the darkest band in the gel. An analysis of the molecular weights of the bands through electrophoretic mobility (Tab. 3) showed the standard Taq having a molecular weight of 115.2 + 14.6 kDa, and the likely band (E) had a molecular weight of 113.4 + 14.3 kDa. There was a distinct distortion in the bands of the SDS page in all lanes with the exception of the standard Taq and the 2011 Taq (Fig. 3). The distortion is of a smile. The overall gel also has a large distortion, but of a frown. It would appear there was a similar protein to D E and F present in all samples, including the 2011 sample. The standard Taq did not contain the bands. Re al Time PCR The Western Blot (Fig. 4) revealed distinct A master mix for PCR was prepared (1x PCR bands; however, there were more than one band in buffer minus Mg, 0.2mM dNTP, 1.5 mM MgCl2, each lane with the exception of the standard Taq. 0.5ÃŽ ¼M forward primer, 0.5ÃŽ ¼m Reverse Primer, 0.1 Two distinct bands were present in 5, Taq, and 2 (b, ng Template DNA and Nuclease-free PCR water). c). The lanes of * and ? contained several bands To PCR tubes, 22.5 ÃŽ ¼L of Master Mix and 2.5 ÃŽ ¼L also. The overall gel also expressed a slight color of Taq sample or the standard Taq were combined, banding along the solvent front edge which is mixed through vortexing and then centrifuged with shown in both Fig. 3 and 4. The 2011 lane did not a Fisher Scientific Accuspin micro 17 just briefly appear to have any Taq present, as no band was using 1.5mL eppendorf tubes with no caps to distinguished. The entire ladder expressed some contain the PCR tube. The Taq samples were antibody activity. prepared in triplicates. 20ÃŽ ¼L of each sampled were The real time-PCR revealed a threshold reached then transferred to a 96-well PCR plate and then at 20 cycles, with the vast majority occurring at 24 sealed. The well was then placed in a BioRad CFX cycles. The melt curve showed an approximate connect Real Time System using the programing of melting temperature of 81 °C (Fig. 7). enzyme activat ion (95 °C, 30 seconds, 1 cycle), 40 The agarose gel electrophoresis revealed one cycles of Denaturation (95 °C, 1 second) and distinct band at approximately 5883.5 base pairs in annealing/extension (60 °C, 5 seconds), with a melt length. The brightest bands, and therefore the curve of (60-95 °C in 0.5 °C intervals, 3 seconds per highest quantities of Taq enzyme were found in the step, 1 cycle). The samples were then analyzed std., 2 and 4. When the base pairs specific activity using AlphaEase FC software. of the enzyme was calculated it was found to be 834.5 + 63.9 bp/min/ÃŽ ¼g of sample, or 3922.3 + 192.9 bp per minute. RESULTS The results of the Bio-Rad assay on the sample of Taq polymerases diluted to 10x and 100x revealed that the 10x dilution was far to concentrated and fell outside the linear curve of the Bio-Rad assay. The retrieval of protein from the Luria broth was found to be 300.8 + 17.7 mg protein per L of Luria broth. These results (Tab. 1) suggest the protein 5 DISCUSSION Through the analysis made through SDS-PAGE, the MW of the standard Taq was found to be 115.2 + 14.6 kDa and 113.4 + 14.3 kDa. This is different from the accepted literature value of 94 kDa (9). Even with error correction, the prot ein did not fall Isolation of Recombinant Taq Polymerase for PCR within the range of the accepted literature value. In total, the two proteins differ by 23% and 21% without error correction, or 21.2 kDa and 19.4 kDa respectively. In comparison to one another, the two bands have essentially the same molecular weight, indicating whatever error occured in the gel was equivalent on both the standard and the isolated Taq. One explanation for the difference in the molecular weights may be explained through the quantitiy of protein used. The darkest and thickest band ( E, fig. 4) likely belongs to the Taq protein. To get a more defined band, a dilution would be effective in making a higher resolution band (12). The amount of protein isolated per volume of Luria broth was determined to be 300.8 + 17.7 mg per L of Luria Broth. Quite obviosuly, there are issues both with the heating of the gel, and distortion of the bands into â€Å"smiles†. The distoration of the gel likely was caused by unequal heati ng of the gel causing the center of the gel to be hotter than the peripheries, as the walls of the apparatus act as heat sinks (13). The uneven heating can be removed by switching to a lower voltage for a longer period of time (12). The distortion of the protein bands within the individual lanes produced a smile structure. The distortion was likely caused by either an overloading of proteins, which can be solved by dilution of the protein sample, or was due to salt conditions of the loading sample. This step could be fixed through extra steps of dialysis to decrease salt content of the loading sample. (14). One final issue with the SDS-PAGE gel was the distance between bands. The target molecular weight was near 100 kDa, so the concentration of the gel could be decreased to allow for a higher resolution of the higher molecular weight proteins, or allowed to run for a longer period of time (14). A purity assessment of the isolated Taq enzyme can be made through the SDS-PAGE gel (fig. 2). Distinct banding occurs in ten different bands on the Taq lane, with 9 being distinct from Taq protein (E). This highlights that there were infact multiple proteins still present in the Taq solution. This would indicate that the heat shock portion of the methods was insufficient in denaturing all of the proteins in the E. coli, allowing for precipitation upon salting out. This is based on the extra protein banding only occuring for the Taq polymerases prepared for this experiment. A factor that could have also played a role was the incubation at 75 °C was continually 6 interrupted through the need to shake the reaction vessel thereby lowering the temperature of the solution. This was due to mechanical difficulties of the equipment. It would be best to find a working New Brunswick Scientific-Innova 40 incubator shaker series to improve the protein isolation. To decrease the protein impurities, an increased heat cycle could be implemented, as Taq is thermostable at 75 °C, and could sustain structure at that temperature for long durations (7). The ammonium sulfate salting out would be mor e efficent after an increased heat cycle as even fewer native state proteins would remain (10). Another method to decrease impurities would be to add a purification step using another specific property of Taq polymerase. This could be the isoelectric point. This could be done through ion exchange columnsor isoelectric focusing (12). The extra isolation step would significantly decrease the impurities, and increase the specific activity per mg of protein of sample.The impurities were likely a result of other proteins present in E. coli bacteria lysate that were relatively thermostable, as those proteins would be most probable (9). The isolation of Taq can be confirmed through the Western Blotting and PCR reactions (Fig. 4-7), as a distinct band in the Western Blot, and measureable amplicon replication in the PCR and rt-PCR. In the standard of Taq of the Western blot (Fig. 4) there is a distinct band. The same band in the channel containing the isolated Taq can be seen. The band occurs in the same relative vicinity as the Taq molecular weight band in the SDS-PAGE (Fig. 2) so would fit best fit the Taq enzyme. The banding of the blot shows a common band across all lanes that line up with the standard Taq, emphasizing the isolation of Taq. There is a hesitation in confirmation of Taq due to the extra protein banding in the prepared fractions, as these bands were not seen in the standard Taq. The banding would suggest proteins transferred from the gel to the membrane and was still able to bind to the primary antibody or secondary antibody. There are various possible explanations for this. First and foremost, the banding occurred in areas wherever protein was present (ladder and lanes). This would indicate lack of specificity in the primary antibody which is intended to only find full sequence Taq and bind to it (15, 16). Another problem may be due to lack of blocking solution binding to the membrane, or Isolation of Recombinant Taq Polymerase for PCR excessive washing removing blocking solution from the membrane. A final possible explanation may be binding of the secondary antibody to membr ane bound proteins with the exception of casein (the blocking protein used) (15, 17). Antibody specificity can be corrected by finding a new antibody, lack of blocking simply requires longer blocking periods or increased blocking solution concentration, and washing can be minimized to see resultant effect on the membrane. Each of the possible problems with the Western Blot would have to be tested by altering the procedure used above by one method (washing, antibody, blocking solution). The PCR results show template replication through thermocycling, which indicates the presence of thermostable DNA polymerases in the PCR tube. From this, it can be conferred that Taq polymerase was indeed isolated. Further confirmation could be made through further purification of Taq. This could be done through 2-D SDS-PAGE vs Isoelectric point electrophoresis using the isoelectric point of Taq and using the bands emphasized as Taq, and a lower concentration gel (12). Another method would be to analyze the gel bands through other methods such as mass spectropscopy or NMR (18). There wa s distinct differences between three sets of Taq polymerases: the standard, the sample prepared in the previous year, and the sample produced in this experiment. Most distinctly the proteins differ with respect to SDS-PAGE gels. Quite obviously, the purest of the enzymes was the standard Taq, followed by the 2011 sample, and the sample prepared in this experiment. The sample prepared through this experiment had a high amount of a salt concentration and resulted in distorted bands, along with numerous other proteins present in the sample. The enzymes also differed with respect to the Western Blot (Fig. 4). The 2011 sample failed to return 2 ° antibody response, indicating lack of Taq polymerase, or lack of primary antibody binding, while the standard and experimental sample both had representive banding. There may have been excessive blocking or drying of the lane containing the 2011 Taq, as the SDS-PAGE shows a representive band in the region of Taq, that is the darkest band in the lane (15). The protein concentrations as determined through the Bio-Rad assay (Tab. 1, Fig. 1) returned 7 drastically different results. The two protein concentrations differed by 2x concentration. The easiest explanation of thi s result is the 10x dilution was insufficient in reducing the absorbance to within the standard curve. Due to the absorbances being above the standard curve, the results are invalid, as the region in which the curve is linear is up to 0.5mg/mL (19). The 100 x dilution returned a result of 1.88 + 0.11 mg/mL. This coroborates the SDS-PAGE findings as the protein was not excessively overloading the lane. The SDS-PAGE could have been further diluted, but the concentration used was sufficient for the purposes of the experiment. In an analysis of the PCR results (fig. 7), the brightest fluorescence bands occurred in the std., 2 and (4/Taq) lanes. This would indicate the highest activities occuring in these lanes. When compared to the western blot, the darkest banding of regions of Taq (5,?,*) returned the bands with less fluorescence. This result shows that the amount of enzyme may inhibit the PCR reaction as the the bands with the highest recoveries returned the lowest fluorescence. With an assessment of the basepair length, reaction time, and amount of enzyme used, an approximately activi ty of 834.5 + 63.9 bp/min/ÃŽ ¼g of protein, or 3922.3 + 192.9 bp per minute. In comparison to the literature values of the protein, this is slightly above the 60 base pairs per second value, however, that was at 70  °C (7). The rt-PCR returned a consistent melting temperature of 81 °C (Fig. 6)for all amplicon samples indicating the lack of a primer-dimer formation. Threshold was initially reached at 20 cycles (Fig. 5), which an RFU value of approximately 9000. This indicated a high activity of the taq polymerase used, at least above 1.25 Units (20). Both PCR assays agree with one another. There was no primer dimer formation noted on the agarose gel, or the melt curve analysis. There was a high activity of the enzyme sample isolated as found through the bpmin-1 and cycle # of reaching threshold, however, between the two assays, the rt-PCR has the significant advantage of time, and no electrophoresis required. Currently, Taq is widely available and would likely be cheaper to simply purchase commercially. This experiment does however outline a method for thermostable protein isolation which could be used for the more recent and more valuable thermostable enzymes (Pyrococcus furiosus Polymerase) which Is olation of Recombinant Taq Polymerase for PCR are superior to Taq in both thermostability, and error rate due to proofreading ability (21). Overall, the purpose of the experiment was met. Taq was indeed isolated from a culture of recombinant E. coli. This was confirmed through the Western Blotting, and thermostable DNA activity in the PCR and rt-PCR. The purity was assessed and found to be below that of the methods used by Engelke et al., 1990. The purity could be increased through use of a cation exchange column (9). The length of heat denaturation and an automatic heat controlled shaker would help to remove excess proteins and improve purity. The length of dialysis time would need to be increased for less band distortion in SDS-PAGE, and either more selective primary antibody, increased blocking or decreased washing would be required for improved Western Blotting. For further experiments, it is suggested testing the new method modifications, and or implementing recombinant Pyrococcus furiosus Polymerase.