Measuring Binding Constant of Ethidium Bromide (EtBr) to DNA

Measuring Binding Constant of Ethidium Bromide (EtBr) to DNA ABSTRACT: The principle behind this lab experiment was to determine the binding constant of ethidium bromide to DNA using the known concentrations and the calculated value of X obtained from the equation. Ethidium bromide is said to have a high affinity to DNA, therefore, the expected value for the binding constant should be large. Two method were implemented in determining the binding constant of EtBr to DNA. The first method was by inputting the absorbance data using a fixed excel worksheet. The main goal was to manipulate the value in cell I24 to be as low as possible. After several trails and error, the final value of cell I24 was 0.00011316 and logK was 3.99. The second method was by simple manual calculation. The two methods yielded two very different results. By manual calculation the obtained value for K was 37,108.63 M-1, which was indicative that the final EB complex of DNA was larger than that of unbound DNA and unbound EB. The high value of the binding constant (K), demonstrates th at there is a high affinity of ethidium bromide to DNA. This correlates to the expected values of EtBr, which are reported to be in between 104 M to 106 M. INTRODUCTION: DNA plays an important role in biological systems being that it contains hereditary materials that is passed on to generations after generations. DNA contains sequences of specific bases within the DNA strands where it stores genetic information that can be readily replicated (Jeremy M. Berg, 2015). It is this sequence that determines the sequence of RNA and other protein molecules and it also transports most of the activities within the cells. RNA synthesis is a key step in the expression of genetic information (Jeremy M. Berg, 2015, p. 859). DNA is more than just a source of sequence information, but it is also the platform where binding proteins collate. This is an important factor for the development of many clinical drugs. The structure and function of drug targets are the basis for designs of effective and specific inhibitors. Though to be considered effective, the target drugs must bind to the enzymes or receptors with great affinity and specificity. Ethidium bromide, (EtBr), is widely used in many scientific laboratories to study the binding properties of DNA. Ethidium bromide is an aromatic dye that slips in between the base pairs of DNA. This binding of ethidium bromide to DNA is a process called intercalation. During this process, the structure of the DNA is changed and the distance among the base pairs in which it directly binds to increases. Resultant in the overall expansion of the dimension of DNA. Ethidium bromide exhibits diminutive base pair partiality containing moderate binding affinity depending on its ionic strength. The intercalation specificity depends greatly on electrostatic interactions and the formation of Van der Waals interactions between base pairs. It has the capability to bind itself to the hydrophobic interior in the stacked base pairs of DNA. However, Ethidium bromide is known to be a very potent mutagen, which is a chemical agent that can cause mutation in DNA cells and other certain diseases such as cancer. It is called an intercalating agent due to its ability to inhibit cell growth, DNA replication and transcription. In this experiment, we will be measuring the binding constant of the dye, ethidium bromide, using herring testes by method of absorption spectroscopy. Herring testes DNA (htDNA) is a natural DNA used in studies of DNA binding agents that modulate DNA structure and function (Sigma-Aldrich, Inc). This method involves titrating a series of solutions varying in DNA concentrations but with a constant concentration of the intercalating agent, ethidium bromide. By implementing this order of magnitude, it is likely to obtain samples with entirely unbound DNA and DNA that is saturated with ethidium. The solutions will run through the absorbance spectrometer at a wavelength of 480 nm. The absorbance measured will be used to calculate the bound ethidium. To determine the amount of bound ethidium the following expressions below is used, given that the information of DÕ†¢ and EÕ†¢ are known; where EÕ†¢ is the total ethidium bromide concentration and DÕ†¢ is the total DNA concentration. This information will allow us to calculate the binding constant of ethidium toward DNA. The equilibrium: D + E à ¢Ã¢â‚¬  Ã¢â‚¬â„¢ C D = DNA E = Ethidium Bromide C = Complex K = [C] / [D][E](1) Solve for K: K = x/[EÕ†¢ x][DÕ†¢ x](2) Kx2 x(K[DÕ†¢] + K[EÕ†¢] + 1) +K[DÕ†¢][EÕ†¢] = 0 (3) Determination of the amount of bound ethidium (amount of complex, C): Aobs = ÃŽÂ µb x + ÃŽÂ µf [EÕ†¢ x](4) ÃŽÂ µb (480 nm) = 2,497 M-1 cm-1 ÃŽÂ µf (480 nm) = 5,600 M-1 cm-1 EXPERIMENTAL PROCEDURES: MATERIALS: 2 mM DNA (bp) stock solution with BPES buffer 2  µM DNA (bp) stock solution with BPES buffer 10  µM stock solution of Ethidium Bromide Micropipettes Microcentrifuge tubes Absorbance spectrometer PROCEDURES: Determine the volume of stock DNA solution and the amount of buffer needed for each of the nineteen samples before proceeding. *Refer to data tables* In the microcentrifuge tubes, make nineteen 1 mL DNA solutions by diluting from the 2 mM, and the 2  µM DNA stock solutions with BPES buffer. Then add 10  µL of ethidium bromide to the nineteen prepared solutions. Mix well and measure the absorbance at 480 nm. Record the absorbance for each of the nineteen solutions and use the information to calculate the binding constant K. RESULTS: DATA TABLE: Absorbance at 480 nm -Log[bp] [DNA(bp)] Volume 2 mM DNA Solution (in  µL) Volume 2  µM DNA Solution (in  µL) Volume of BPES buffer (in  µL) 0.018 3.0 0.001 500.0 XXXXXX 500.0 0.023 3.3 5.01187ÃÆ'-10-4 251.0 XXXXXX 749.0 0.024 3.7 1.99526ÃÆ'-10-4 100.0 XXXXXX 900.0 0.032 4.0 0.0001 50.0 XXXXXX 950.0 0.032 4.3 5.01187ÃÆ'-10-5 25.0 XXXXXX 975.0 0.029 4.7 1.99526ÃÆ'-10-5 10.0 XXXXXX 990.0 0.032 5.0 0.00001 5.0 XXXXXX 995.0 0.031 5.3 5.01187 ÃÆ'-10-6 3.0 XXXXXX 997.0 0.030 5.7 1.99526ÃÆ'-10-6 1.0 XXXXXX 999.0 0.032 6.0 0.000001 XXXXXX 500.0 500.0 0.033 6.3 5.01187ÃÆ'-10-7 XXXXXX 251.0 749.0 0.033 6.7 2.51189ÃÆ'-10-7 XXXXXX 100.0 900.0 0.032 7.0 0.0000001 XXXXXX 50.0 950.0 0.032 7.3 5.01187ÃÆ'-10-8 XXXXXX 25.0 975.0 0.034 7.7 1.99526ÃÆ'-10-8 XXXXXX 10.0 990.0 0.035 8.0 0.00000001 XXXXXX 5.0 995.0 0.035 8.3 5.01187ÃÆ'-10-9 XXXXXX 2.5 997.5 0.033 8.7 2.51189ÃÆ'-10-9 XXXXXX 1.0 999.0 0.030 9.0 0.000000001 XXXXXX 0.5 999.5 DATA GRAPH: CALCULATIONS: Sample Calculation for K: Using sample #4 Known Information: Aobs = ÃŽÂ µb x + ÃŽÂ µf [EÕ†¢ x] Aobs = 0.032 ÃŽÂ µb (480 nm) = 2,497 M-1 cm-1 [E]initial = 10  µM = 1.0 ÃÆ'-10-5 M ÃŽÂ µf (480 nm) = 5,600 M-1 cm-1 [DNA(bp)]initial = log(bp) = -4.0 = 1.0 ÃÆ'-10-4 M Solve for X: Aobs = ÃŽÂ µb x + ÃŽÂ µf [EÕ†¢ x] 0.032 = (2497 M-1cm-1)x + (5600 M-1cm-1)[(1.0 ÃÆ'-10-5 M) x] x = 7.73943 ÃÆ'-10-6 M Plug in value of x to solve for K: K = x/[EÕ†¢ x][DÕ†¢ x] K = (7.739 ÃÆ'-10-6 M)à ·[( 1.0 ÃÆ'-10-5 M) (7.739 ÃÆ'-10-6 M)][( 1.0 ÃÆ'-10-4 M) (7.739 ÃÆ'-10-6 M)] K = (7.73943 ÃÆ'-10-6 M) à · [2.26057 ÃÆ'-10-6 M][9.22606 ÃÆ'-10-5 M] K = (7.73943 ÃÆ'-10-6 M) à · (2.0856148 ÃÆ'-10-10 M2) K = 37108.63 M-1 K = 3.71 ÃÆ'-104 M-1 FINAL EXCEL WORKSHEET: After refinement DISCUSSION: The principle behind this lab experiment was to determine the binding constant of ethidium bromide to DNA using the known concentrations and the calculated value of X obtained from the equation. Ethidium bromide is said to have a high affinity to DNA, therefore, the expected value for the binding constant should be large. However, two methods were used to obtain the value K (binding constant). The first method was by using excel worksheet and inputting our data. The main goal was to manipulate the value in cell I24 to be as low as possible. After several trails and error, the final value of cell I24 was 0.00011316 and logK was 3.99, if you take the antilog of that value K would equal to approx. 9772.37, which is low in comparison to method two which was done by manual calculation. By manual calculation the obtained value for K was 37,108.63 M-1 which indicates that the final EB complex of DNA was larger than that of unbound DNA and unbound EB. The high value of the binding constant ( K), demonstrates that there is a high affinity of ethidium bromide to DNA. This correlates to the expected values of EtBr, which are reported to be in between 104 M to 106 M. Using a buffer that does not contain added NaCl, such as BPE, will have different results than that of a buffer with NaCl, such as BPES. The BPE buffer will yield a lower binding constant than that measured in BPES buffer. It is well known that the interaction within the process of intercalation is driven by electrostatic factors and à Ã¢â€š ¬-stacking with the bases (lab manual). The electrostatic binding of ethidium bromide to DNA has a preference to binding to the phosphate backbone the DNA strand. The DNA-ligand binding is salt-dependent due to the counter-ion release thats carried out during binding. This is indicative that the salt component in the buffer demonstrates a relatively greater stability in DNA due to its preference to the binding site within the GC-rich DNA region. With that said, it is apparent that an outlying positive charge is essential for intercalation. The positive charge on the intercalation diminishes as the aromatic system increases. Like ethidium bromide, Actinomycin D is another known intercalator with a high affinity to DNA. Though the two differ by means of binding sites. Actinomycin D intercalates at GC sites, which indicates that the two intercalators would not compete with one another at the exact binding sites. Therefore, by adding Actinomycin to a solution of herring testes DNA Ethidium bromide, resulting in two different results. An example of the plot is shown below: The plot is simply an example of what it might look like. There are a lot of variables that must be considered in choosing the proper intercalator. Factors to consider are concentrations of solutions and DNA, buffers, whether its a low salt concentration or high salt concentration buffer. The difference in buffers could possibly yield two very different results. Another factor to consider is the magnitude of the absorbance. All these factors combined could hinder the final outcome, so it is hard to conclude exactly how the actinomycin D would react in combination with ethidium bromide to DNA within this experiment. REFERENCES: Jeremy M. Berg, J. L. (2015). Biochemistry 8th ed. Kate Ahr Parker. Eva M. Talavera, Pablo Guerrero, Francisco Ocana, and Jose M. Alvarez-Pez, Photophysical and Direct Determination of Binding Constants of Ethidium Bromide Complexed to E. coli DNA, Appl. Spectrosc. 56, 362-369 (2002) Fuller, W., and M. J. Waring. 1964. A Molecular model for the interaction of ethidium bromide with deoxyribonucleic acid. Ber. Bunsen Ges. Phys. Chem. 68:805-808. Qiao C, Bi S, Sun Y, Song D, Zhang H, Zhou W (2008) Study of interactions of anthraquinones with DNA using ethidium bromide as a fluorescence probe. Spectrochim Acta A 70: 136-143 Graves, D. E., C. L. Watkins, and L. W. Yielding. 1981. Ethidium bromide and its photoreactive analogues: spectroscopic analysis of deoxyribonucleic acid binding properties. Biochemistry. 20:1887-1892. [PubMed] Suh D, Chaires J B (1995) Criteria for the mode of binding of DNA binding agents. Bioorg Mediclin Chem 3(6): 723-728

Nathaniel Hawthorne’s The Birth-Mark, Raymond Carver’s Cathedral, and R

Nathaniel Hawthorne’s â€Å"The Birth-Mark†, Raymond Carver’s â€Å"Cathedral†, and Randall Kenan’s â€Å"The Foundations of the Earth† illustrate how arrogance undermines knowledge and individual power and humility enhances those qualities. In each story, characters with parochial worldviews encounter people who challenge them to change. Other perspectives are available if they are able to let go of their superior attitudes. For example, Hawthorne’s protagonist, Aylmer, believes he has the ability and right to create perfection. He views a birthmark on his wife, Georgiana, as evidence of a flaw that must be removed no matter what the cost. His assistant, Aminadab, (an earthy alter-ego) remarks, â€Å"If she were my wife, I’d never part with that birthmark† (Hawthorne 531). He does not say, â€Å"I’d let it be† or â€Å"I’d tolerate it†, but rather â€Å"I’d never part with it.† This interpretation is so antithetical to Aylmer’s that it cries for inquiry. â€Å"What is it that you are think ing, Aminadab?† or â€Å"What is it about this birthmark that I find so ugly that you would treasure?† Aylmer does not ask these questions. Arrogance shuts him down. One needs humility in order to consider alternative points of view. New ideas do not enter Aylmer’s mind and he does not develop. His arrogance culminates in the death of Georgiana. In the other two stories, however, the characters mature by humbly opening to diverse perspectives, thus gaining knowledge and individual power. 1 Raymond Carver’s short story â€Å"Cathedral† opens with a narrator whose wife has invited a blind friend to spend the night. The narrator depersonalizes the man right off the bat and repeatedly throughout the story by referring to him, not by name, but as â€Å"the blind man† (Carver 513). He admits that hi... ...h. On the other hand, arrogance stifles one’s growth by shutting out different perspectives. One is left with nothing except what one started with; one’s mind becomes a closed box of stifling inflexibility or a Pandora’s box of anger and blame. Sometimes arrogance leads to a fate like the one Georgiana and Aylmer experienced in Hawthorne’s short story. Works Cited Hawthorne, Nathaniel. "The Birth-Mark." Reading Literature and Writing Argument. Ed. Missy James and Alan Merickel. Fourth ed. Boston: Longman, 2011. 527-38. Print. Carver, Raymond. â€Å"Cathedral.† Reading Literature and Writing Argument. Ed. Missy James and Alan Merickel. Fourth ed. Boston: Longman, 2011. 513-23. Print. Kenan, Randall. "The Foundations of the Earth." Reading Literature and Writing Argument. Ed. Missy James and Alan Merickel. Fourth ed. Boston: Longman, 2011. 149-61. Print.

Who Made the Mistake

NAME: ZEENAT AMEEN MOHAMMED ID NO: 7401 COURSE NAME: CROSS CULTURAL COMMUNICATION TOPIC: WHO MADE A SERIOUS MISTAKE? LECTURER: DR. SHARON CASE STUDY: WHO MADE A SERIOUS MISTAKE? 3. 1IDENTIFYING THE FACTS OF THE CASE: The facts of the case are: * Jean safari was investigating a serious error made by a Japanese worker at the Japanese subsidiary of a US multinational. * A component had been inserted upside down, and the entire batch has been pulled out of production to be reworked. * The cost of repairing is on the high side. * The director claimed not to know who was responsible for the damage. The group did not tell him the specific woman involved. * The whole group has accepted responsibility of the event. * The woman concerned tried to resign from work but her coworkers coax her back again, the groups is aware that she is new and they did not help her enough or look out for her or see that she was properly trained. * The entire group decided to take responsibility and apologize to j ean. 3. 2 STATING THE PROBLEM The problem in this case is that Jean needs to find the culprit who fixed the component upside down. 3. 3 SETTING THE OBJECTIVESThe objectives of this case are to find out the culprit and for the culprit to be punished in order to avoid future reputation of the same event. 3. 4 IDENTIFYING THE AREAS OF CONSIDERATION IN THIS CASE The problem in this case is that jean wants to know the culprit in order for her to punish the person involved to avoid any similar case in the future, but the plant manager regards this as not important if the culprit has realized her mistake, but on the other hand, Jean Safari thinks its important for her to find out the person, and take an action against her.Jean Safari is a UNIVERSALIST and believes in INDIVIDUALISM also meaning she is concerned about the rules of the firm and the person involved should be punished individually, but the plant manager and the remaining workers are PARTICULARIST and they believe in COMMUNATALI SM, they believe in the relationship they have with one another and they work as a group which makes them COMMUNTALIST in nature.UNIVERSALIST: In Universalist culture, the rules apply equally to the whole †universe† of members, regardless of relationships General rules, codes, values and standards †What is good and right can be defined and always applied† In a Universal society such as the U. S. , rules and contracts are developed which can apply in any situation. There is a belief that what is good or true can be discovered, defined, and applied to every situation.Because of their Universalist approach, in a business situation Americans will want to rely on a contract to communicate the terms of an agreement and to define the relationship between the parties. PARTICULARISM: Particularism is based on logic of the heart and human friendship. China is a particularistic culture where people look at relationships and circumstances in a specific situation to decid e what is right. For the Chinese, the legal contract communicates a starting point for an agreement. As circumstances change so too should the terms of the agreement.For the Chinese, the situation and the particular individuals involved are what define relationship. INDIVIDUALISM: Individualism is a theory maintaining the political and economic independence of the individual and stressing individual initiative, action, and interests also conduct or practice guided by such a theory. Individualism places great value on self-reliance, on privacy, and on mutual respect. Negatively, it embraces opposition to authority and to all manner of controls over the individual, especially when exercised by the state.As a theory of human nature, individualism holds that the interests of the normal adult are best served by allowing him maximum freedom and responsibility for choosing his objectives and the means for obtaining them. COMMUNATALISM: Communitarianism is an ideology that emphasizes the co nnection between the individual and the community. That community may be the family unit, but it can also be understood in a far wider sense of personal interaction, of geographical location, or of shared history. 3. 5 PROPOSING THREE ALTERNATIVE COURSE OF ACTIONS ST ALTERNATIVE: The first alternative would be to use PUNISHMENT (SUSPENSION) because if the culprit is being suspended from work he would not be paid for those days that he wont be around because it would be considered he is not working for the main time he would not be around this would help in disciplining of other workers in the organization and it would avoid further mistakes and lack of concentration and new employees would be watched thoroughly and all the existing workers would help the new ones and get them on track to do the new job. nd ALTERNATIVE: The second alternative could be CONSIDERATION, the culprit could be considered so as to allow the worker with the impression he would not repeat the same mistake agai n.The advantage of this is the worker would be happy to work in the firm knowing that the firm considered him and this also would motivate him/her and other coworkers but also consideration might also have a negative effect, if the workers know that they are always considered when they make a mistake every worker would make a mistake and would expect to be considered employees will develop a sense of rebellion in response to punishment rather than seeing it as a deterrent to a particular activity. rd ALTERNATIVE: The third alternative should be WARNING a culprit could be seriously warned not to attempt doing such offence again and if he repeats it a serious action would be taken against him/her I this way the culprits and the other co workers would be very careful in their dealings in the firm, the disadvantage of this might be, some workers would not adhere to the rules and regulations of the firm knowing that the highest thing that can happen would be a warning letter. 3. 6 CHOOSI NG AND RECOMMENDING THE BEST SOLUTIONThe best solution to this case is WARNING and SUSPENSION because if the culprit is warned she would not repeat such offence again in future since it would be clearly stated that if the offence is done again a serious action would be taken, if the culprit is punished this would make other workers scared and some might even leave the organization which would be at the risk of the organization in general, and also if the worker is suspended this would bring a huge difference in the attitude of workers in the organization since they know that a serious action would be taken against them therefore they would be more careful when they are doing the job.BIBLIOGRAPHY bukchin, m. (2012, october 25). http://www. via-web. de/universalism-versus-particularism/. Retrieved march 22, 2013, from http://www. via-web. de/universalism-versus-particularism/: http://www. via-web. de/universalism-versus-particularism/ media, d. (2012, september 5). http://www. meez. c om/community. dm? furl=http%3A//forums. meez. com/forums/serious-topics/6221608-individualism-vs-communalism. html. Retrieved march 22, 2013, from http://www. meez. com/community. dm? furl=http%3A//forums. meez. com/forums/serious-topics/6221608-individualism-vs-communalism. html: http://www. meez. com/community. dm? furl=http%3A//forums. meez. om/forums/serious-topics/6221608-individualism-vs-communalism. html mitchel, c. (2011, august 6). http://www. theaustralian. com. au/national-affairs/opinion/individualism-versus-communalism/story-e6frgd0x-1226109346928. Retrieved march 22, 2013, from http://www. theaustralian. com. au/national-affairs/opinion/individualism-versus-communalism/story-e6frgd0x-1226109346928: http://www. theaustralian. com. au/national-affairs/opinion/individualism-versus-communalism/story-e6frgd0x-1226109346928 webstar, m. (2013, february 3). http://www. merriam-webster. com/dictionary/particularism. Retrieved march 22, 2013, from http://www. merriam-webster. co m/dictionary/particularism:

I Was Told A Sad And Scary Event Happened At Tianjin...

I was told a sad and scary event happened in my alma mater. When I heard this news firstly, I could not believe that breaking news. This event caught my eyes and caused my deep reflection about the cause of this tragedy. The 19 years old freshman girl who was study at Tianjin Normal University of Elementary Education has suicide on April 10th, 2015, because she carried the hepatitis B virus. She was tested positive for the virus during a blood donation on Dec 6th, 2014. Her parents and classmates said, she was a funny, aggressive and optimistic girl at the beginning of her campus life. When her classmates and teachers were told she carried virus, she was asked to live alone in a single room. In fact, she had three roommates before she moved out. I must say a truth that this virus can only be transmitted by exposure to infectious blood or through sexual contact. She became negative and disappointed for the attitude of her friends and teachers. Her friends would not touch her things and stay or talk with her. Her parents worried about her health and negative emotion, so they required one year suspension of schooling, then they went back to their hometown. When she asked to back to the univers ity, one of the school leaders has asked her parents to get a certificate stating that an HBV carrier doesn t affect the school. She started to feel despair and ruthlessly. She chose to end her life when she was 19 years old. Her parents said, â€Å"the school s requirement