Any Kind Checks Cashed, Inc. v. Talcott Essay

According to the UCC, a holder in due course is a holder who takes an instrument for value in good faith and devoid of notice of assured claims, as well as defenses on the instrument. It is imperative in the case of ‘Any Kind Checks Cashed, Inc. v. Talcott’ to determine if the holder of the instrument acted in good faith, in fair dealing as is compulsory in order to be considered the holder in due course. According to the Commercial Law Article 3-103 (a) (4); good faith refers to sincerity or honesty in fact and the adherence to logical commercial standards of fair dealing (Twomey, D., & Jennings, M.). Apart from this, the court upholds the trial court finding that Any Kind did not act in good faith when cashing a check for $10,000. However, Any Kind had been in good faith in later on cashing one more check for $5,700. I agree that ‘Talcott’ was responsible for the $5,700 even despite the fact that he was illegally persuaded to issue a check (Legale .com). The procedures in place at Any Kind Checks Cashed, Inc.states that a supervisor that has the power as well as authority to approve checks over $2,000. However, the supervisor should have been more cautious due to the unusual amount of the check of $10,00.00. When the second check of $5,700.00 was presented the check cashing company had been in contact with Talcott getting his verbal approval for cashing a check. As a result, Any Kind Check Cashed Inc. satisfied the good faith prerequisites for a holder in due course. In reviewing if, the tellers actions where in accordance with good faith and acted reasonable within the commercial standards for accepting and processing the check. The fact is that the clerk did take action and question the intent of the check of $10,000.00 by calling the maker of the check Mr. Talcott, but was not able to reach him. As a result  of not reaching Mr. Talcott the, the supervisor relied on her judgment and experience to make her decision to cash the check. Using her judgment coupled with appeared to be evidence the FedEx envelope showing that the FedEx was sent from Mr. Talcott the supervisor was acting in good faith but not in accordance with a the reasonable commercial code. There should have been some level of suspicion that someone would pay a 500.00 fee to cash a check and not go to his or her bank and collect the full amount of the 10,000.00. To a reasonable person this type of behaviour may raise a red flag as to the desperation of the person (payee) to cash such a large check. The check cashing store should have verified with the issuing bank to ensure that there was enough monies in the account to cover the check and verify that the check is good (no stop payment was issued). When Mr. Guarino presented a second check to the check cashing store for $5,700.00, the store reached out and spoke with Mr. Talcott asking him to verify the check for $5,700.00, which he verified. The check cashing store never mentioned the first check of $10,000.00. Perhaps they presumed that since Mr, Talcott approved the second check that and never said anything about the first one being of issue it may have seemed as a nonissue. If Any Kind processed the $10,000 check with proper caution and procedures beyond making a phone call and not getting an answer from the maker of the check. Any Kind should have contacted the bank the check was drawn on to verify that there was no issue with the check and not rely on experience and a FedEx cover. In order to ensure that they and preserved their status of the holder in due course status. The courts determined that Any Kind was not the holder in due course due to the manner in which they did not ensure that the check was valid before cashing and processing it. I agree with the court’s decision in finding that Any Kind is not the HDC for the $10,000.00 since they were really negligent in the handling and processing of the check. I agree, with the court’s ruling in favour that Any Kind is the HDC for the $5,700.0. Since ‘Any Kind’handled the process in good faith and within accordance with reasonable commercial standards according to the UCC 3-419[3] (Legale .com). References Cornell Law: Uniform Commercial Code. Retrieved Dec 30, 2014. http://www.law.cornell.edu/ucc/3/3-419 â€Å"Federal Reserve Bank: Regulation and compliance guide.† Federalreserve.gov. Retrieved Dec. 28, 2014. http://www.federalreserve.gov/bankinforeg/reglisting.htm ANY KIND CHECKS CASHED, INC. v. TALCOTT | Leagle.com. (n.d.). Retrieved from http://www.leagle.com/decision/2002990830So2d160_1973.xml_br Twomey, D., & Jennings, M. (2014). Kinds of Instruments, Parties, and Negotiability. Business Law: Principles For Today’s Commercial Environment (4th edition ed., pp. 567- 568). Mason: Cengage learning.

Aquinas’ and St. Anselm’s Proofs of the Existence of God

It is generally understood that people that are nonbelievers in God would never believe in the philosophies of the believers with regard to His existence. Nevertheless, there have been a number of important philosophers who have sought to prove the existence of the Almighty by thought alone. According to these philosophers, the presence of God may be understood by reason. Throughout the ages of humanity, believers have claimed to have felt the presence of God. All the same, there is no room for personal proofs in the philosophy of religion. St. Thomas Aquinas was a believer in God, who proposed five ways to know God. The first way of proving God’s existence, according to the philosopher, is by means of observing motion; that is, everything in movement throughout the universe should prove to us that there is a Prime Mover of all things. This way of proving God’s existence is similar to the argument of the creationists who assert that everything that we observe must have a Creator, just as all things that are man-made have originators. On a similar note, the second way of knowing God according to St. Thomas Aquinas is by understanding that all effects have causes; and so, there must be a First Cause of everything. The third way of proving the existence of God is by observing that everything in the universe is â€Å"possible to be and not to be;† so therefore, there must be an Essential Existence without which the existence and nonexistence of other things are meaningless (The Summa Theologica of St. Thomas Aquinas, 1920). This proof is intricately tied to the earlier proofs, including the current argument of the creationists. St. Thomas Aquinas describes it thus: The third way is taken from possibility and necessity, and runs thus. We find in nature things that are possible to be and not to be, since they are found to be generated, and to orrupt, and consequently, they are possible to be and not to be. But it is impossible for these always to exist, for that which is possible not to be at some time is not. Therefore, if everything is possible not to be, then at one time there could have been nothing in existence. Now if this were true, even now there would be nothing in existence, because that which does not exist only begins to exist by something already existing. Therefore, if at one time nothing was in existence, it would have been impossible for anything to have begun to exist; and thus ven now nothing would be in existence — which is absurd. Therefore, not all beings are merely possible, but there must exist something the existence of which is necessary†¦ This all men speak of as God (The Summa Theologica of St. Thomas Aquinas). St. Thomas Aquinas also writes that everything that is necessary must have been made necessary by something else. However, there must be something that is necessary in and of itself. Furthermore, this entity of necessity must cause other entities to believe in its necessity. This is the meaning of God, according to the philosopher (The Summa Theologica of St. Thomas Aquinas). The fourth proof proffered by St. Thomas Aquinas is similar to Plato’s concept of Forms. The philosopher believes that there is a ranking in place throughout the universe (â€Å"Thomas Aquinas and the Five Ways,† 2000). One person may be better than the others. Given that we compare attributes of people and things to rank them reveals that there must be â€Å"something which is truest, something best, something noblest and, consequently, something which is uttermost being;† in short, something that is Supreme in comparison with the rest (The Summa Theologica of St. Thomas Aquinas). The fifth way of proving the existence of God is exactly the same as the argument of creationists in our times. St. Thomas Aquinas discussed intelligent design with the philosophy that everything in the universe serves a special, intelligent purpose that has been designed by the Creator. This proof is based on the assumption that everything around us is essentially unintelligent – except God, of course (The Summa Theologica of St. Thomas Aquinas). In fact, all five proofs of God’s existence proffered by St. Thomas Aquinas may be said to revolve around the intelligent design argument. St. Anselm was another believer like St. Thomas Aquinas who proved the existence of God by reason alone. Yet, the ontological argument proffered by St. Anselm is the simplest. Instead of five proofs of God’s existence, St. Anselm offered only one, that is, God is that beyond which there is no greatness that we can conceive of (â€Å"St. Anselm’s Ontological Argument,† 2008). But, St. Anselm’s argument also rests on five logical points. The following is the argument with its corollaries: (1) God is that than which no greater can be conceived; 2) If God is that than which no greater can be conceived then there is nothing greater than God that can be imagined; Therefore: (3) There is nothing greater than God that can be imagined; (4) If God does not exist then there is something greater than God that can be imagined. ; Therefore: (5) God exists (â€Å"St. Anselm’s Ontological Argument†). The gist of the above argument is the very definition of God’s existence, according to St. Anselm. This definition – â€Å"God is that than which no greater can be conceived† – is different from all proofs of St. Thomas Aquinas (â€Å"St. Anselm’s Ontological Argument†). Moreover, it is a reasonable proof, similar to St. Thomas Aquinas’ statements about God’s existence. After all, it is easy to suppose that God must be that beyond which we can conceive of no greatness. Besides, nobody in the history of humanity has ever claimed to be greater than God. Even if someone were to claim that he or she is greater than God, it would be impossible for the majority of humans to believe in the claim, seeing that God would not compete with the claimer as in a debate. With our limited perceptions, we are unable to see God, and neither could we imagine something or somebody that must be greater than God. If we cannot find anything that is greater than God, the Almighty must be Real, according to St. Anselm. Even so, the essential argument of the philosopher does not answer the beliefs of the atheists that perhaps there is no greatness in the universe, as everything is equal. Similarly, St. Thomas Aquinas’ arguments are refuted by the atheists who proffer proofs of the evolution theory of Charles Darwin to contradict the existence of God. According to the atheists, the proofs of St. Thomas Aquinas and the ontological argument of St. Anselm could be refuted because they are based on basic beliefs of the people rather than universal facts. The truth that St. Thomas Aquinas believes in the First Cause of everything means nothing for the disbelievers in God, who might claim that there is nothing to precede anything except in the evolutionary cycle that everything must go through, that is, things evolve one from another without a Prime Mover or First Cause. As a matter of fact, there is no argument to bridge the gap between the claims of believers and disbelievers in God. For as long as we know the difference between truth and falsehood, there would be arguments. The claims of each group are refuted by those of the other. It may very well be that these arguments and refutations would continue for as long as there is humanity on our planet. Seeing that God cannot be seen, and neither does He support the believing philosophers by speaking out aloud to all the rest that these philosophers are correct in their belief systems – it is possible to refute the godly thoughts of St. Thomas Aquinas and St. Anselm until the end of time. Conversely, those who believe in their philosophies and do not doubt could be ones that have felt the presence of God beyond reason. In fact, believers in God may suppose that it is impossible to know God by reason alone. Given that truth cannot be turned into falsehood by argument alone, there must be a singular truth about the existence of God nevertheless. What is the truth? – In the discipline of philosophy, it is the fact that everything in the universe is not known to be confined to the realm of reason. For the believing philosophers, therefore, God is a Reason beyond reason.

American Revolutionary War General Essay

The American Revolution was the course by which numerous American colonies became an autonomous nation. It involved novel ideas grounded on republicanism and needed success in a long battle with Britain. Following the fighting that raged for nearly a year, the United States declared independence in 1776, as an autonomous country and established a coalition with France that leveled the naval and military might of the two nations. The fundamental cause was the British government’s refusal to permit Americans voice their concerns in setting duties. During the pre-revolutionary period, some notable events such as the Braddock’s expedition influenced the revolutionary path. The Braddock’s expedition commenced in 1755 in Pennsylvania . Among the volunteers during the expedition was Daniel Morgan who is considered one of the most victorious field leaders during the American Revolution. The tales of this renowned patriot and the regiments that operated under his command in the Boston siege, assault of Quebec, the obliteration of Burgoyne’s forces at Saratoga as well as his devastating conquer of Banastre Tarleton cannot be forgotten. Not much is known concerning the Daniel Morgan’s early life. It is generally thought that he was born in New Jersey in the year 1736 . His early life was signified by a rough frontiersmen outlook. He left original home when he was a child and established in Winchester, Virginia. Morgan was remarkably strong and a tireless, hardworking worker. He started by employing himself out but within a very short period of time earned sufficient confidence from his boss to be in charge of the boss’s sawmill. Although Morgan would sink into debts severally, he managed to save sufficient money to establish an autonomous wagon. Generally, Morgan was a prosperous captain, militia, farmer and a respectable citizen. When in 1775, the Continental Congress sanctioned the recruitment of two firms of Virginia riflemen, Morgan was the unchallenged choice from his Virginia County to recruit and head one of them. Early Career Daniel Morgan had an unconquerable spirit together with rapid wit made him a favorite amongst the people and before long most of the colleagues from Berryville and Winchester looked upon him as their leader . An agitated and high-spirited individual, he abandoned home in his early teens and established in Virginia. The muscular man was repeatedly in problems with the laws for affraying in taverns and failing to pay card debts and liquor. As a trucker, he accompanied the unfortunate Braddock’s expedition. His career started his career by working as a planter but was later given a job to work as a trucker for a wagon. Within a period of six months, Daniel had saved sufficient cash to purchase own team and cart and became a trucker under his own management on the Great Wagon Road . This road started in Philadelphia, traversing via Lancaster, shifted southward to Maryland, Frederick, traversed through Winchester, crossed the middle and eastern sections of Virginia Valley ending up in North Carolina. Morgan’s first hand experience of North Carolina and southwest Virginia added to his eye-catching defeat of Tarleton and the following idiotic tactics of Cornwallis to arrest him resulting to the British conquer at Yorktown as the American Revolution ended . Braddock’s Expedition The war of Monongahela commenced in 1755, near the location of contemporary-day Braddock, Pennsylvania. After it ended, over five hundred British forces lay dead and approximately five hundred were wounded . Only three hundred managed to escape the menace uninjured including George Washington . Edward Braddock, a British commander had been fatally wounded and succumbed to injuries several days later. Braddock’s men suffered badly from the initial stages of the war. The Tactics used by Indians were supremely appropriate for woodland battle. Battle between the French and British on the border implied additional work for Daniel who volunteered ferrying materials for the British forces. The rival assertions of England and France for the rich valleys southwest of Alleghenies had started to create slaughter. The French had already conquered the England fort located on the Allegheny Rivers and Monongahela and Washington had attempted to repossess it only to admit defeat at Great Meadows. Colonel Braddock was dispatched from Britain to conquer the tracts on the western side of the mountains. He brought a force comprising a collection of artillery and two regiments. There was need to procure transportation and Daniel volunteered his services. In addition to the regulars, Braddock also enjoyed the company of several men and camp followers. The forces were gathered at Cumberland pending the Braddock’s arrival. The aim was to proceed but the mission was delayed for several days while teams and wagons were found to carry the equipments and baggage. Once the regiment was in motion the journey was extremely slow. The roads were dominated by creeks and swamps and the terrains rough. Braddock made a decision to walk ahead with one thousand two hundred men and to abandon the artillery and baggage with Colonel Dunbar. Dunbar’s forces set camp at Great Meadows, the dawdlers started arriving with information concerning Braddock’s defeat. Fear and panic spread throughout the entire camp at the imagination that the enemy might attack. The following day most of the troops went back home together with most of the teams and wagons, abandoning the helpless and wounded. Due to the nature of Daniel’s work, he was among those in the wagon trains when Braddock’s forces were assaulted and nearly eliminated by an Indian and French alliance; instead of materials, Daniel found himself ferrying wounded soldiers . The condition on the Virginia frontier and Pennsylvania was ominous. The Braddock’s defeat left the border open for the murdering and robbing of the border populace. At a period when condition demanded all men to be available to safeguard the defenseless population, Dunbar and his troops left for Philadelphia. The Virginia’s administration met the condition with swiftness and energetically raised an extra force of sixteen firms under Washington to protect the frontier. Washington assigned forces at different places along the frontier. Morgan was assigned the responsibility of ferrying supplies along the defense lines. This was an extremely risky task as a trucker and was regularly exposed to serious dangers from the loitering foe. Morgan escaped death narrowly no several occasions. Experience with British Forces In the year 1756, Daniel experienced a dreadful disagreement with a lieutenant from Britain . The officer had been offended by something Daniel had articulated and after abusing him verbally; he hit him with the edge of his blade. Daniel’s instantaneous right cross hit the officer meaningless knocking him cold. The defiant act led to speedy military justice. The British laws were excessive on this form of crime and Daniel was accorded five hundred strokes, the harshest penalty possible. Such punishments were neither uncommon for the British forces nor was it unusual for men to pass away under the stroke. He obtained all of the punishment except one stroke. Daniel was too strong to pass away, but the familiarity left his body a piece of hamburger and bone . Once the wounds healed over, nonetheless, he acquired a priceless visual aid that proved important to him severally. It was articulated that the penalty would have murdered a slighter man. Until this period Daniel had not commanded anyone. He had helped Braddock and in return got wrongly trodden for his endeavors. The battle frenzied on and extended to the bottom of the blue hills. Fort Louden and Fort Cumberland were attacked by the foe standing just two hundred miles away from Daniel’s dwelling in Winchester. The militia was called to Fort Garrison and Daniel headed the forces towards the fort and commanded to for some period. Although none of his position is documented, it is widely assumed that he commanded the garrison. Shortly after his arrival, a body of Indians and French attacked Fort Edwards. Owing to Daniel’s leadership and bravery, the assault was halted. The battalion sallied forward and surpassed their enemies wounding and killing numerous Indians as the escaped in all directions. In 1758, General Forbes, the leader of the British battalion planned to assault Fort Duquesne; Daniel was suggested by leading officers of Virginia for the captaincy post. Governor Dinwiddie rejected the recommendation and only an ensign’s charge was obtainable. Daniel accepted the charge and was positioned at different forts beside the border in 1758 . Devastating Attack Daniel was dispatched from these forts with a company of two defense force . He was assaulted by a group of Indians and French. The defense forces were blasted and fell right away off the horsebacks. Daniel was stroke by a gunshot that pierced via his neck, scraping one part of his neck-bone, and then passing via the mouth . The gunshot removed all of Daniel’s teeth, but surprising enough didn’t harm the jawbone. In other words, Daniel’s head had been shot. Although wounded terribly, Daniel never fell from the horseback. The blood oozed effortlessly out of the gunshot wound and he was vulnerably weak, yet in such circumstances he was capable of keeping his feelings long enough to run away from additional harm. The horse he rode on panicked at first and froze, But Daniel seized her neck in an attempt to spurn her on . Incredibly, the horse curved and headed back to the fort. The Indians, assuming Daniel was fatally wounded, descended upon the defense forces searching for scalps. Daniel, also assuming he was just about to perish, speeded the horse to escape from his chasers to avoid mangling of his body. Daniel’s horse arrived at the fort when he was insensible. He remained in a critical condition for a long period of time, but with judicious treatment and good care, he recovered. Daniel later went back to Fredrick where his manly and bravery behavior had accorded him a high standing amongst his folks. Experience as a Lieutenant The Indian and French War ended and it seemed as if Daniel would get a chance to lead a nonviolent live. But following the peace treaty Pontiac’s battle broke out, an onslaught that destroyed the whole Western Frontier. Virginia reacted by recalling militia into active service to assist the existing battalion already stationed on the border. The forces were put under the authority of Colonel Stevens. Daniel was posted as a lieutenant in the regiment. In a crucial battle, the Indians were conquered and forced to implement a peace agreement without Daniel participating in the battle. The band of soldiers was disbanded and therefore ended the war. Leader of County Militia In 1771, the governor of Virginia, William nelson commissioned Daniel as Captain of Frederick County’s militia . This was a period of great extension on Virginia border, a development which disappointed Indian populace. Great carnages were happening in places such as Kentucky and Virginia’s populace feared that the trouble would spread to Virginia quickly. Their fears were confirmed and atrocities were committed by a band of settlers, instigating Dunmore’s War. Governor of Virginia, Lord Dunmore, was confident to deal with the issue himself. He commanded a big battalion from the northern region to be organized and four brigades of volunteers and militia to be arranged in the Southern regions. While staffing for these regiments was being executed, a defense force was recruited to position itself between the border and the settlements under McDonald command. It was under the command of McDonald that Daniel took the leadership. He recruited several men and proceeded to Wheeling in Virginia to link up with McDonald. Major McDonald was initially requested to assume a defensive location, but decided to launch offenses due to lack of arrangements with the Indians. Daniel’s forces rapidly pushed the Indians from their locations forcing them to retreat. An Experienced Fighter By the year 1775 Daniel was an assessed and country fighter. He was capable of fighting using Indians methods of fighting and was proficient in using Pennsylvania gun, a long, thin tool of great accuracy and range. Not amazingly, when the Congress commanded the recruitment of ten firms of border riflemen to work as light infantry, Daniel was selected as a captain. After the U. S revolutionary War started at the skirmish of Concord and Lexington, the Congress established the famous Continental Army. The Congress gathered and called for creation of rifle firms from colonies to assist the Boston Siege and General Washington was named the commander of the army. It permitted the recruitment of twenty thousand men into the army. Congress also required ten firms of riflemen to be called into service in Virginia, Maryland and Pennsylvania states. Among the companies provided by Virginia State, Daniel Morgan was chosen as the leader of one rifle firm by the commission of Frederick County through an undisputed vote . Daniel’s first significant assignment surfaced in 1775, when worked in Benedict Arnold’s voyage that attacked Canada . Exposed to the waist and sporting Indian breechlout and leggings, Morgan led the expedition. During the assault, he took momentary command Montogomery’s murder. He battled valiantly against the foe until ultimately overpowered by larger numbers and forced to surrender. Although Daniel spent eight months in an English detention prior to exchange, his notable achievements at Quebec gave him merited acknowledgment. He was endorsed for the post of colonel and provided with a special force of light infantry comprising five hundred selected backwoodsmen. Achievements during Revolution Saratoga War Daniels’ light forces had its best moments in the Saratoga battle of 1777, when he rushed to help the northern army, then refuting the southward campaign from Canada of General Burgoyne . Burgoyne had witnessed his forces repulsed at Bennington and Oswego and his weapons run precariously thin in New York. During the battle, American colonel Horatio permitted Burgoyne to exploit his supplies in unsuccessful probes. Morgan was used by Gates to annoy and delay the enemy. The rifleman, utilizing their woodland knowledge effectively killed many redcoats. Soon enclosed by Gate’s forces and bands of militiamen, Burgoyne surrendered at Saratoga. As other attacks in the south were being evaluated, Britain’s standing among the colonies was tarnished both militarily and politically with enemies as well as friends. Morgan went back to Washington’s forces with splendor and immediately joined army politics. He believed his achievements would earn him a larger command as well as promotion particularly because a novel light infantry was being formed. However, the Congress felt that Virginians fielded sufficient Generals and offered the authority to a Pennsylvanian, Anthony Wayne. Cowpens War After a long period of inactivity, Morgan chose to accomplish his stand at Cowpens after the American grounds brightened in the South with a novel leader, Nathanael Greene who dispatched him into South Carolina . Confident to eliminate Daniel before attacking the higher south, Cornwallis dispatched Tarleton to pursue him. Daniel’s assumed advantage of Tarleton’s behavior for rapid action and his disregard for the inexperienced militia and the accuracy and longer range of Virginia riflemen. The gunmen were located to the frontline while both the regulars and militia remained behind. The first units were required to withdraw upon being threatened thus provoking a premature attack. During the campaign, Morgan famed himself as a leader of troops who nevertheless sustained the ordinary touch and maintained their secret. Typical soldiers toiled harder and shoved themselves a little bit further when Daniel was supervising them. The approach achieved enormous results; as the English troops neared, the American troops reloaded their guns. When the enemy got close they fired in their faces. Within a very short time, Tarleton’s one thousand and seventy six men got wounded, one hundred and ten killed and eight hundred and thirty captured . Although Tarleton managed to escape, all his equipments and supplies were captured by the Americans. Daniel’s cunning strategy is widely viewed to be the strategic masterwork of the battle and the most effectively executed envelopment of contemporary military history . Conclusion Braddock’s conquer was indeed one of the drastic events of the period before American Revolution. It left population settlements in Virginia and Pennsylvania susceptible to Indian and French invasion. Braddock’s expedition also improved Daniel Morgan’s standing and assisted helped him to be the sensible choice for an officer of Riflemen units in the American Revolution. Finally, the expedition was a significant chapter to the long battle with the Indian forces for the administration of the western Pennsylvania and Ohio valley. Saratoga War was also a significant success, won when United States required success in the most awful way and played an integral role in influencing other governments such as French administration to formally join the battle to aid the besieged colonies. The coalition with France became pivotal in maintaining American Revolutionary military resistance and resolve Bibliography Cassell, Frank. The Braddock Expedition of 1755: Catastrophe in the Wilderness, n. d, < (29 July 2009) Cheaney, Janie. Daniel Morgan, 1998, < http://jrshelby. com/kimocowp/morgan. htm> (29 July 2009). Chapel, Hill. Daniel Morgan: Revolutionary Rifleman. North Carolina: University of North Carolina Press, 1979. Daily, Allen. History of the Descendants of David Morgan, 2000, (29 July 2009). Frassett, James. Revolutionary War, 2000, (29 July 2009) Jacob Cushing, A Sermon Preached at Lexington, April 20th 1778. Boston: Powars & Willis, 1776. John Archdale, â€Å"A New Description of that Fertile and Pleasant Province of Carolina,† Narratives of Early Carolina, (1911): 277-313. Richard Price, A Discourse on the Love of Our Country . . . Commemorating the Revolution in Great Britain. London: Edward E. Powars, 1789.

Dell Marketing Case Study Example | Topics and Well Written Essays - 500 words

Dell Marketing - Case Study Example The Chinese market had grown to be the largest in the region from a volume standpoint, and as with each of the company's international channels posed its own set of unique challenges. The market leader domestically was Lenovo, a company which had an extensive small-to-medium scale business model appropriated for the country. In order to compete with Lenovo, for starters Dell tripled its production capacity by shoring up its operations in Xiamen. Bear in mind that for any sustainable competitive advantage to be achieved by the company in the region, the strategic plan must be closely adaptable to the pertinent market segments. Although opening its doors has resulted an a hitherto unseen westernization of the country, the smaller cities remain highly industrial in nature. Hence, the market demographic remained strictly lower income, most of whom were not comfortable ordering over the Internet. Dell's strategic answer to this was to open numerous offices across Chinese cities in deference to their direct sales business model. And as mentioned earlier, the unfamiliarity with E-commerce led Dell to make extensive use of toll-free numbers to cater to the less sophisticated business segment.

The July plotters of 1944 were not courageous men whose purpose was Essay

The July plotters of 1944 were not courageous men whose purpose was honourable but reckless men who acted out of their own self interest How valid is this assessment of the July Plot of 1944 - Essay Example storted political circumstances of the country in Germany wiped out the war situation and Hitler’s public interaction has reduced in monthly visit in Berlin. The leaders of the conspiracy included Field Marshal Erwin Rommel, General Ludwig Beck, Lieutenant Colonel Claus, and Count Schenk von Stauffenberg, The conspirators like Himmler and Stauffenberg were suspicious and they had their own personal intentions than the unified goal. Stauffenberg took the responsibility to carry out the assassination attempt. During the time of German Campaign against Soviet Union, Stauffenberg became frustrated with Hitler’s misrule. Beginning of his career Stauffenberg has revealed his loyalty and responsibility to Hitler and to Nazi Germany. But towards the end of 1930s, like others Stauffenberg began to think that Hitler’s misrule pull the nation in to the disaster. Stauffenberg’s plan was to plant a briefcase with the bomb in Hitler’s conference room. Stauffenber g’s words reveal his will power and intention. The article remarks;† In August 1942, he told a friend: â€Å"They are shooting Jews in masses. These crimes must not be allowed to continue† (Peter, 2004 P. 2). After the unsuccessful attempt of July plot Stauffenberg was honored as a courageous warrior who made an unending fight against Hitler’s despotic rule. A dreadful reality is that the purpose of the conspirators has changed and it came to be observed by some of them as achieving the military honors themselves. Most of them have worked under Hitler’s administration in their past. Martin Collier, Philip Pedley state that; â€Å"In Berlin, the conspiracy was paralyzed by indecision and vacillation† (Martin Collier& Philip Pedley, 2005 p. 122) Field Marshal Witzleben was a soldier who participated in the event of July plot 1944. Witzleben and others were found guilty and sentenced to hang. His dreadful death proves that he was a courageous fighter. The following comment about the conspirators underlines

Land Law Question Essay Example | Topics and Well Written Essays - 1000 words

Land Law Question - Essay Example As per UK law there are two sorts of land a) registered and b) unregistered. Each has its own rules and regulations with regard to treansfer of land from one hand to another hand besides enforcement of third party proprietary interests1. The Land Registration Act 2002 which is in vogue came into being on October 13th 2003. New Act has more space for a) number of titles b) over riding interest and c) electronic conveyancing. This has simplified the land transfer system. Hence, buyer needs not to worry about the vendor’s entitlement to deal with the property2. Unlike unregistered land where the purchasor needs to go through the register himself in order to find out whether third party proprietary interest is binding or not. Kinds of Leases The basic leases are four in numbers a) Gross Lease b) Full Service Lease c) Gross Industrial Lease and d) Triple Net Lease. Each landlord has to decide which lease is best suited to him. It is the duty of the attorney to let his clientle (ten ant) know which lease is beneficial to him. In commercial type of real estate lease, interest of stakeholders (Landlord and the Tenant) is ensured. It would be in the best interest of mentioned parties if they chose right type of lease3. Tenancy Agreement In accordance with the Landlord and Tenant Law of UK, tenancy agreement is an agreement that executes between the landlord and the tenant for renting out the premises. By all means, it is a legal contract. It can be in two ways, oral or written. However, the written agreement is more viable option for both the stakeholders, i.e. landlord and the tenant since it provides more concrete evidence and binding in terms of law4. Yes, some of the tenancy terms are implied even if they are not mutually agreed upon. Implied terms indicate rights, obligations and the protective clauses in line with the Residential Tenancies Act currently enforced. The Residential Tenancies Act fully protects the rights of the tenant. If the tenancy agreement includes something mutually agreed upon by the landlord and the tenant, which is not allowed by the said Act, would be binding for either party to comply with it5. Sometimes people are a little bit confused with regard to the meaning of word â€Å"lease†. In fact, the mentioned word refers to the fixed term, say one year or more tenure. This is incorrect. It can be on a month-to-month or week-to-week basis. In order to avoid confusion, the term â€Å"lease† is not used frequently in such transaction. However, in the larger interest of the landlord and the tenant, the term â€Å"tenancy agreement† can be used for the rented property6. Fixed Term Tenancy In a fixed term tenancy, the tenant agrees to have the premises for a certain period of time against mutually agreed amount of rent and the security deposit. Under the mentioned situation, if the period of time ends, the tenancy agreement ends automatically. Here, no notice is required to serve by either party unt il and unless it was specifically incorporated in the tenancy agreement. If the notice period was incorporated in the said agreement, either party is bound to serve the notice accordingly. After the expiration of the fixed term of tenancy, if the tenant does not vacate the rented premises, the tenancy agreement turns into periodic tenancy agreement, according to which, the tenant has to pay the rent as desired by the landlord. Once a tenancy period starts, it

Zeitgeist and architecture Essay Example | Topics and Well Written Essays - 1250 words

Zeitgeist and architecture - Essay Example In respect of the building of choice in this paper, Le unite de Habitation du Marseille form Le Corbusier, It is important to underscore the fact that architectonic space is a syntactic language that attempts to transmit concepts through our most basic instincts. Architecture incorporates the reaction in philosophy and the beliefs of a particular time. Such architectural designs have a direct impact on space which also goes in line with time. From time immemorial art has been part of everyday life of people in various cultural settings. This has been the case with the various aspects of the society ranging from structures, interior design of houses, food arrangement patterns and designing of clothes. Various theoretical concepts have been advanced in respect of the philosophical factors that are taken into consideration when designing various buildings. Architecture is one of the oldest sciences that require in-depth understanding of various factors in respect of the desired structur e. A number of modern towns are experiencing increasing demand for housing with a blend of diverse architectural designs (Mitias, 1999). There are various theoretical concepts that are spoken by the structure itself once it is constructed. Architecture captures the space factor which is increasingly becoming scarce in most residential sites. There have been many cases of setting up buildings in a way that they blend with the topography of the site and the cultural affiliation with the immediate society. Various architecture scientists attest to the fact that the contemporary society strives to spare any little space and maximize the allocated site with an expansive interior and relatively slim exterior. It is common to come across a number of features in many cities which still represent the ancient architecture and are mainly taken through renovation with the building technique kept intact. The cultural affiliation of the people is an important factor in designing various architect ural pieces of art. The ancient time is a reflection of the origin of various concepts and socioeconomic and political concepts that dictated a given way of life amongst the members of the community. This explains the diverse architectural features that are found around the world and the cultural association they have with the given community. It is worth to note that the contemporary architecture considers the serenity of the surroundings and the complexity of the social characteristics of the society. This has been brought about by the increasing sensitivity of people towards colors and their blending (Gargiani, Le Corbusier, Rossellini & Piccolo, 2011). It is important to note that any sensitive architectural creation must incorporate the important influence of image to the public when designing a house. Design of a building says more than words about the prevailing culture, and it explains why some of the ancient buildings have kept under the UN conventions on preservation of cu ltural heritage. The contemporary emergence of globalization is driving urban centers towards over population and a beehive of commercial activities. This requires redefinition of planning the town in terms of structures. Space is becoming a great issue and this explains the research on how the architectures and town planners can come up with beautiful and space saving designs. The increasing competition for space is a matter of great concern among various competing demands of an urban center. There is a need for a revised polychromy of architecture. Ultra-modern art has seen most of the houses designed with vibrant colors and attractive interior design with paints that are water resistant for durability (Mitias, 1999). The adoption of

Technology in classrooms Research Paper Example | Topics and Well Written Essays - 750 words

Technology in classrooms - Research Paper Example Nevertheless, there has been a challenge in trying to get teachers to be comfortable and secure with their technology skills. This is particularly common with practical subjects such as social studies where technology applications would be used for tutoring purposes (Shriner et al., 2010). There has been a categorization of issues, which influence integration of technology into classrooms; they are curricular, social, environmental and personal aspects; moreover, environmental aspects refer to elements such as administrative support by the school, hardware and software. The social aspect focuses on the significance of communities, to support technology in classrooms. The personal aspect relates to the individual viewpoint on technology by teachers and their willingness to adapt it. Lastly, curricular aspects relate to use of technology in specific tutorial settings. That is, how the learning materials are linked with the course plans (Lih-Juan, 2007). It is widely agreeable among dif ferent social communities and educators that educational practices need to prepare students to flourish in the dynamic technological world, as it has gained acceptance. Hence, teachers are required to embrace technology and be willing to familiarize themselves with it. This will not only boost their confidence but also improve their teaching experiences as when applied effectively, technology enhances student learning, promotes shared learning and motivates them to seek more knowledge outside the classroom (Hubbel et al., 2012). In a bid to encourage teachers to familiarize themselves with technology advances in the classroom, various learning institutions have asked teachers to view them as a means of further developing themselves professionally; thus, this will advance their skills and knowledge. On the other hand, the learning institutions need to avail the necessary technologies to the teachers and ensure they are up-to-date whilst making their teaching schedules flexible enough for them to take the time to learn (Lih-Juan, 2007). Teaching tools that have come up due to advances in technology As aforementioned, there are the basic teaching tools that have been incorporated: the internet, LCD projectors, smart boards, document cameras and digital cameras. The internet is obviously the most widely used; teachers use it to access Web-based resources. The others offer visual assistance in the classrooms, which is the key in deepening understanding. The LCD projector is mounted in classrooms, for teachers to deliver Power Point presentations, share videos and other activities; moreover, it allows information from a single computer to be usable in a large group setting. Over time, this has proven to be the method that makes students of this generation responsive. The document camera has replaced the old overhead projectors and works hand in hand with the LCD projector. It enables teachers to magnify images and texts from diagrams, charts and textbooks for a bett er view by the students. The smart board enables charts, templates and diagrams to be rearranged, students can participate actively in the lesson and at the end, and it can be printed out as handouts for them. It requires lengthy training to use effectively but because of the eagerness it builds in students, teachers are

Healthcare Essay Example | Topics and Well Written Essays - 250 words - 2

Healthcare - Essay Example We must understand the total package, by understanding their belief, culture. It is necessary to tap into their mental, emotional and physical need. Part of being a One Stop shopping medical facility and to include all of the right individuals from different field and title that is going to work all under the same umbrella to meet the patient care. Therefore, we would be engaging the help of a social worker to make those visits that would be necessary within the patient’s habitat. A community liaison is the key to providing continuing support, that will be providing a sense of accountability. Researchers have proven that when there is a reward at the end of any goal that helps keep the person focused and the goal more attainable. We have established an incentive program, where we would reward the patient for milestones attained, by writing a blurb in our newsletter detailing their journey and how they have done it to give others hope. The patient education would begin upon admission and continue for the duration of the program. It will flow over to the provider’s office and through the community, we believe that an educated patient will be less resistant and easier to help reach their goals. We offer many different options that the patients can take advantage of like group counseling or one on one therapy. In all this cultural factors will be considered to avoid conflicts with the

Culture And Second Language Learning Essay Example for Free

Culture And Second Language Learning Essay Through the conduction of studies, it has been discovered that learning a second language, particularly in a situation where second language learning is given particular importance, requires an immersion into the lingua and cultural societies of the native speakers of the second language that is to be learned (Wan 44-45). This requirement is extremely important to be followed as the immersion on the societal components of the second language of the native speakers helps the learner to obtain more hands-on learning benefits when trying to acquire second language abilities (Wan 45). Literature Review Although numerous language specialists do conclude that culture and social groups exercise great influence on the acquisition of second language abilities, these specialists are still bound to not realize that the components that involve this situation are interconnected and are not distinct entities from each other (Wan 46). It is crucial to understand that learning a second language involves factors that cannot exist without the existence of other factors (Wan 47). Most recently, it has become a trend to become immersed in the culture of the native speakers of the second language through the use of the internet (Wan 47). Numerous second language learners browse the web in order to get immersed into the native speakers’ culture (Wan 47). Numerous second language learners can definitely learn through the utilization of this method as the web has got a lot of information from around the world to offer to the browsers (Wan 47). Methodology And Data Analysis Result of the surveys participated in by English language learner students: 3 1. Methodology: They have had difficulty forming friendships due to language barriers. Data Analysis: A little over 65 % of these people have had trouble forming friendships with English native speakers in the United States due to language differences. It is often uncomfortable to form a friendship with people whom you often encounter difficulty in conversing with. An interesting conversation turns sour when you have difficulty conversing with someone. The people who face this difficulty with English native speakers often become lonely while living in the United States if they are not able to find friends that share the same language as them. 2. Methodology: They have had difficulty achieving their academic goals than they would have if their instruction at school were conducted in their native language. Data Analysis: Although non-native speakers of English who are proficient in English are the only non-English speaker foreign students who are admitted for formal academic studies at schools in America, English proficient non-native English speakers also still encounter difficulty in their academic studies than they would have if the instruction was conducted in their own language. This is such the case as the mentioned students still are not used to the English language. These students most usually converse with their circle of influence in their native language, hence, this situation causes them to become awkward in utilizing the English language in their academic studies as well. The most objective advice that can be recommended from this situation is for the students to use the English language often even when conversing with non-native English speakers who form their circle of influence. Or, it would be better if these students would be able to find native English speaker friends who would be willing to practice conversing with 4 them in English on a regular basis. It would also be helpful for these students to practice sharpening their English language writing skills through regularly utilizing writing drills. 3. Methodology: Language barriers often make the students feel a longing to go home to their country. Data Analysis: Numerous students who experience difficulty learning English in the United States are usually not able to adjust to their social environment in the country. As a result they often feel that they are social outcasts and feel that they don’t belong in the community. Because of this situation that they may get into, they may become inclined to go back to their home country. 4. Methodology: Language barriers may have the tendency to make the students inculcate a lower self-esteem. Data Analysis: The students feel that they are not smart enough as they are not able to efficiently learn a second language. This dampens their spirits of good self-esteem. 5. Methodology: These students become the targets of discrimination pranks in the United States. Data Analysis: Numerous of these students become the victims of prank jokes just because of their limited English language abilities. They are seen as vulnerable group of people as they create a self-image of being dumb or â€Å"not smart enough† due to their limited English language abilities. 6. Methodology: These students become too dependent on an English proficient relative, family member or friend while in the United States. Data Analysis: This becomes the situation as the students may not even be able to convey simple information to strangers (e. g. , giving orders at a restaurant) due to their limited 5 English language abilities. The mentioned family member, relative or friend may eventually feel that these students are becoming a burden on them as time goes on. 7. Methodology: Numerous students are reluctant to learn English as a second language because they feel that learning it would cause them to somehow exhibit disloyalty to their own language and culture. Data Analysis: It now a common trend that people do somehow paint a picture on other people as being â€Å"colonially influenced† if they try to learn a second language. This causes these second language learners to become self-conscious when trying to learn a second language. If these people become too self-conscious to learn a second language, they may also give up the pursuance of a goal to move to another country and other goals that come with it. 8. Numerous people abandon their culture altogether and adapt the culture of the native speakers of the second language that they are learning. Learning a second language ultimately also causes one to get immersed in the culture in which that language belongs. Therefore, there are times that the culture of the learners gets totally abandoned as these learners try to learn a second language. This situation is sometimes viewed negatively by others. Results Learning a second language definitely has its ups and downs. The learners would just have to weigh in whether the advantages outweigh the disadvantages as a basis in making their decision to learn a second language. However, we can definitely see that as long as the teaching of the second language is executed properly, numerous students are able to learn the language efficiently and utilize their second language fluency in worthwhile activities and 6 and professions. Discussions Three questions need to be answered in order for specialists to improve second language teaching and learning (Conference Probes Second Language 1). These questions are the following (Conference Probes Second Language 1): 1. What are the strategies on research that need to be executed in the implementation of second language education in America (Conference Probes Second Language 1)? 2. What are the strategies on research on the execution of second language education in the United States that need to be improvised (Conference Probes Second Language 1)? 3. What are these research strategies that are deemed to be effective (Conference Probes Second Language 1)? Reading, writing and speaking fluency of a second language does not come automatically if one has acquired extensive knowledge on the language usage (Swiderski 4). A culture’s main foundation is the official language that comes with it (Swiderski 6). When one first learns a language, he/she automatically first learns its corresponding culture (Swiderski 6). A language mirrors its corresponding culture (Swiderski 6). However, the language and the culture are not exactly similar in every ways (Swiderski 6). Because when learning a language one learns all about its usage, its interconnected component, culture, is also being learned during the process of learning the language (Swiderski 6). Therefore, language and culture are still 7 Regarded as separate entities (Swiderski 6). We can really see that a language tells about what kind of culture it evolves in (Swiderski 13). For example, a classroom where an English language learning class is held comprises of styles and settings that tells about the culture of the country where the school that houses the classroom is located (Swiderski 13). Significant differences in the way that the English language is used among the native and non-native speakers of English are considerably noticeable (Lucas 59). However, there are some questions that arise from this conclusion (Lucas 73). These questions are the following (Lucas 73): 1. Do the students make requests to their professors directly or indirectly (Lucas 73)? 2. Is politeness evident in the way that the students make the requests (Lucas 73)? Conclusions Charles Hutchison, Lan Quach and Greg Wiggan wrote in their article entitled, â€Å"The Interface of Global Migrations, Local English Language Learning And Identity Transmutations Of The Immigrant Academician,† English language instructors who experience teaching students belonging to different cultures, experience the following culture shock situations: 1. Problems in differences in teaching and learning. 2. Problems in differences in curriculum and assessment. In the article entitled, â€Å"The Interface Of Global Migrations, Local English Language Learning And Identity Transmutations Of The Immigrant 8 Academician,† using the social-constructivist (and cognition) theories as references, instructors of English language classes experience changes in the way they view language through the following ways: 1. They may be forced to conform to the students and their circle of influences’ viewpoints and theories. 2. They may experience communication barriers. Furthermore, the authors state in the article that the English language instructors come to realize who they really are and their vocation objectives due to the following reasons: 1. They shape what types of people their students become and their students’ self-esteem. 2. They struggle in helping their students realize their own identity as a minority in the United States. 9 Works Cited Hutchison, Charles, Quach, Lan Wiggan, Greg. â€Å"The Interface of Global Migrations, Local English Language Learning And Identity Transmutations Of The Immigrant Academician. † Forum On Public Policy: A Journal Of The Oxford Round Table (2006). Lucas, Sigrun. â€Å"Students Writing Emails To Faculty: An Examination Of E-Politeness Among Native And Non-Native Speakers Of English. † Language, Learning Technology 11 (2007): 59, 73. Swiderski, Richard M. Teaching Language, Learning, Culture. Westport: Bergin Garvey, 1993. Wan, Shun. â€Å"Second Language Socialization In A Bilingual Chat Room: Global And Local Considerations. † Language, Learning Technology 8 (2004): 44-47. â€Å"Conference Probes Second Language Research Needs. † Reading Today August 2001.

Puc18 Plasmid Engineering

Puc18 Plasmid Engineering Abstract The objective of the experiment was to engineer a pUC18 plasmid so that it contained a kanomycin resistance gene in its multiple cloning site and to transform it into cells. The kanomycin resistance gene was obtained from a pKAN plasmid. The desired plasmid was constructed by digesting pUC18 and pKAN with the same restriction enzymes,(BamHI and HindIII) and religating the products to give the engineered pUC18. The created plasmid was then transformed into E.coli strains DH5ÃŽ ±. The strains that contained the engineered plasmid were selected using two methods of selection. According to the indirect method of selection the percentage of competent cells transformed with the plasmids was 0.063% which is a low number. According to the direct method of selection on the other hand no cells were transformed. In conclusion even though some colonies with the engineered plasmids were obtained the percentage of cells transformed was very low. Also, the indirect method of selection gives better results for selection of desired strains. Introduction Bacteria can carry antibiotic resistance genes either in their chromosomes or extrachromosomally in phage or a plasmid(Hausner and de Jong 2010). B-galactosidase is an enzyme involved into the cleavage of lactose into glucose and galactose and is encoded by the lac Z gene of the lac operon.(Glick et al 2010) The lac operon is prevented from being transcribed through repression of the lac promoter. Activation of this promoter can be done by the addition of lactose or isopropyl-ÃŽ ²-D-thiogalactopyranoside(IPTG) to the medium. Lactose and IPTG simply prevent binding of the lac repressor(the product of the Lac I gene) to the promoter. (Glick et al 2010) In the following experiment plasmids pUC18 and pKAN are used to provide the genes to be transformed into bacteria. pUC18 is 2686 base pairs(bp) long and contains a bacterial origin of replication, an ampicillin resistance gene, a lacI gene, a segment of the lac Z gene encoding part of B-galactosidase(which breaks down X-gal) and a multiple cloning sequence(MCS) that is within the lac Z gene.(Glick et al 2010) The lac Z gene encoded by the plasmid is part of the B-galactosidase protein which complements a gene carried by the Escheria. coli chromosomally thus forming a functional B-galactosidase.(Glick et al 2010) If a DNA segment is cloned in the MCS then the lacZ gene will be interrupted and will not give rise to a functional protein. If that occurs then the Bacteria transformed with the plasmid will not break down5-bromo-4-chloro-3-indolyl-ÃŽ ²-D-ÃŽ ²-galactosidase( X-gal) present in the plates. When X-gal is broken down by ÃŽ ²-galactosidase it turns blue whereas when it is not bro ken down it stays white. This color differentiation is a way to tell if there has been any DNA incorporated in the MCS of pUC18. Finally in order for the ÃŽ ²-galactosidase in pUC18 to be transcribed, IPTG has to be present in the medium so that the lac operon can be induced.(Glick et al 2010) pKAN plasmids can serve as sources for the kanomycin resistance gene. In the following experiment the kanaomycin resistance gene will be inserted in the MCS of pUC18. pKAN contains an origin of replication, a kanomycin resistance gene and multiple restriction sites.(Hausner and de Jong 2010) More importantly it contains only one BamHI and HindIII recognition sites in the whole plasmid which flank the kanomycin resistance gene.(Hausner and de Jong) This allows researchers to cut out the antibiotic resistance gene by simply using BamHI and HindIII producing only two fragments of DNA: the gene and the rest of the plasmid. Once experimenters have inserted the pKAN gene into the MCS of pUC18 and transformed the E.coli strains they need a way to select for the desired plasmid. There are two methods to select for the desired those colonies: the direct method and the indirect method. The direct selection method involves spread plating transformed strains into plates containing both the antibiotic ampicillin and kanomycin. (Hausner and de Jong 2010) Since the pUC18 plasmid confers amplicillin resistance(Glick et al 2010) and the kan gene confers kanomycin resistance (Hausner and de Jong 2010) then only the cells that contain Puc18 with the kanomycin resistance gene should be able to grow in these plates. The indirect method on the other hand is a two step selection process. In the first step the transformed strains are plated onto LB plates containing ampicillin and X-gal.(Hausner and de Jong 2010) Only the cells that have up-taken pUC18 will grow since they will be resistant to ampicillin. Furthermore cell s that contain pUC18 with inserted DNA in the MCS will produce white colonies since they cant produce a functional ÃŽ ²-galactosidase. Cells that give rise to blue colonies will have up-taken pUC18 without any DNA inserted in their MCS since they are able to break down X-Gal. (Glick et al 2010) To select the cells with pUC18 containing the kanomycin resistance gene the white colonies are plated in plates containing kanomycin. Only the cells that have the kanomycin resistance gene in their pUC18 will grow.(Hausner and de Jong 2010) The objectives of the following experiment include the construction of a pUC18 plasmid containing the kanomycin resistance gene in the MCS, the transformation of that plasmid into the E.coli DH5ÃŽ ± cells and the selection of the cells containing the engineered plasmid. If both pUC18 and pKAN plasmids are digested with BamHI and HindIII and the digests are ligated then a plasmid which contains both kanomycin and ampicillin resistance genes should be produced; consequently cells transformed with the engineered plasmid should be resistant to both antibiotics. Materials and Methods Plasmid extraction and plasmid engineering pUC18 and pKAN plasmids were extracted from the DH5ÃŽ ± and MM294 E.coli strains respectively using a DNA isolation kit as described by (Hausner and de Jong 2010). Confirmation for proper extraction was done through agarose gel electrophoresis by running the extracted DNA in a 0.7% gel at 100V for 1 hour. The gene containing kanomycin resistance from pKAN was cloned into pUC18. The restriction digests to do the cloning were prepared as described in Table 2 in (Hausner and de Jong 2010). After plasmid digestion the kanomycin resistance gene was inserted into the multiple cloning sequence of pUC18 in a ligation reaction using the enzyme ligase and the reaction was allowed to go to completion for 24 hours at room temperature. The ligation reactions were set up according to table 3 in (Hausner and de Jong 2010) E.coli transformation and strain selection E.coli strain DH5ÃŽ ± was sub-cultured for 1 hour at 37Â °C. The cells were then made competent by washing them in 10mM CaCl. Next cells were transformed with three different combinations of plasmids. The set of cells in tube 1 was transformed with uncut pUC18 DNA. The set of cells in tube 2 was transformed with cut pUC18. Cells in tube 3 were transformed with pUC18 containing the cloned pKAN resistance and finally cells in tube 4 were transformed with just water as a negative control. The transformation procedure has been described in (Hausner and de Jong 2010). Transformed cells from all tubes were spread plated onto LB+carb+X-gal plates for indirect selection. Furthermore cells from tube 3 were plated onto LB+carb+ kan plates for direct selection of cells containing pUC18 with the insert from pKAN. To determine the density of competent cells cells dilutions of , and were prepared. The two highest dilutions were plated onto LB plates. All the plates were incubated at 37Â °C and they were allowed to grow for ~24 hours. After the colonies had grown on plates plate they were counted and their numbers were recorded. White and blue colonies from the LB+carb+X-gal plates were then streaked onto LB + kan plates to obtain the colonies that had the kanomycin resistance gene incorporated in the MCS. For more information on the procedure refer to Experiments in Biotechnology Laboratory Manual (Hausner and de Jong 2010) Results Extraction of plasmids from E.coli strains Figure 1 contains the image of the 0.7% agarose gel in which the isolated plasmids Puc18 and pKAN were run to check for product. As it can be seen in lane 1 a lot of Puc18 was extracted from the DH5ÃŽ ± strain. Less plasmid DNA was collected for pKAN from the MM294 strain since the band in lane 2 is of much weaker intensity. There is more than one band in lane two. The additional bands represent additional plasmids isolated from the bacteria. Calculation of Competent cell density Table 1 shows the dilutions performed on the competent cells in order to calculate their cell density. It also shows the number of colonies on the plates that were spread plated with dilution 2 and dilution 3. The results for the dilution were not used for cell density calculation since less than 30 colonies grew on the plate. Dilution was used to calculate the cell density because the number of colonies was between 30 and 300. Indirect method of selection Cells plated from tubes 2 and 3 were used to calculate the % of transformed cells. Every colony represents a single transformed cell since it can be assumed the every colony has arisen from a single cell. Furthermore for tube 3 since five plates were spread plated the percentage of the transformed cells was obtained by using the average amount of colonies for all five plates. Calculation the percentage of transformed cells in tube 2: %of transformed cells= x 100 =0.0045% of cells transformed Calculation of transformed cells in tube 3 Average for blue colonies: = 58.6 ≈ 59 blue colonies Average for white colonies = 11.4 ≈ 11colonies Total number of colonies = 59 blue colonies + 11 blue colonies = 70 colonies in total Both blue and white colonies from tube 3 represent transformed cells since they both up-took plasmid DNA whether it was just pUC18 or pUC18+kanomycin resistance gene. Therefore since every colony came from a single cell there were 70 cells in total that were transformed from 100Â µl of media spread plated in each plate. % of transformed cells in tube 3: %of transformed cells= x 100 =0.063% of cells transformed Direct selection of clones containing the kanomycin gene: No colonies grew on LB + carb + kan plates. That means that there were no cells that were transformed with the engineered plasmid. Furthermore an accurate number for % of transformed cell could not have been calculated even if cells had grown in these plates. That is because this selection method takes into account only the cells that were trasformend with pUC18 which contained the kanomycin resistance gene and not the cells that were transformed with only pUC18. Discussion Isolation of plasmids from cells The optimal results for the gel would have been to see one strong band at ~2.7 kb representing pUC18 and one strong band at 4.2 kb which represents pKAN. For the pKAN lane there is more than one band seen. Those bands represent different sized plasmids that were also isolated from the cell. Since there was no DNA ladder on the gel it cannot be concluded what plasmid the lanes represent but the only thing that can be concluded is that there was plasmid DNA isolated from both the DH5ÃŽ ± and the MM294 strains which most likely was pUC18 and pKAN. In order to conclude whether pUC18 and pKAN plasmids were isolated from the bacteria the students should be provided next time with a DNA ladder in order to determine the sizes of the lanes. Indirect selection method The cells from tube 1 were transformed with un-digested pUC18. The cells from this tube represented a positive control for transformation. The colonies in the plates were all blue and they were too many to count. The reason for the high number of colonies was that these cells were transformed with undigested plasmids which are all stable and all allow bacteria to carry information extrachromosomally, making the transformation percentage of competent cells very high. All the cells from tube 1 produce blue colonies. That is because they all had a functional B-galactisidase since no genes were cloned into the multiple cloning site located within the lacZ gene. The cells from tube 2 were transformed with digested pUC18 plasmid. The cells from this tube represented a negative control for kanomycin resistance gene cloning. Tube 2 gave rise to very few colonies in comparison to tube 1 because the cells in tube 2 were transformed with unstable DNA. pUC18 had been previously digested with HinDIII and BamHI and a lot of plasmid did not re-ligate and for that reason the DNA was unstable. Since the DNA was unstable it was not able to maintain the ampicillin resistance gene in bacteria and consequently the strains were not able to grow in carbonicillin plates. As a result the number of percent transformed cells was as low as 0.0045%. The cells from tube 4 were transformed with sterile water i.e no DNA. These cells represented the negative control for transformation. Because no DNA was inserted in them none of the cells contained the ampicillin resistance gene and as expected none grew in the plates containing carbomicillin. The cells from tube 3 were transformed using pUC18 that contained insertion on the MCS as well as pUC18 that didnt. All five plates that were spread plated with E.coli from tube 3 contained blue colonies as well as white ones. The reason for the color difference is that the blue colonies contained a functional ÃŽ ²-galactosidase whereas the white ones didnt. The functional ÃŽ ²-galactosidase in the blue colonies was due to the fact that no DNA was inserted in the MCS to interrupt the lacZ gene. The white colonies on the other hand did not contain a functional ÃŽ ²-galactosidase since they had a DNA insertion in their multiple cloning site, which interrupted the lacZ gene. Consequently they could not break down X-gal. However just because they had a DNA insertion in their MCS it did not mean that they contained the kanomycin resistance gene. They might have contained the rest of the pKAN plasmid. As a result the white colonies needed to be streaked into plates that selected for kanomy cin resistance. If the cells then grew on LB + Kan plates and they also originated from white colonies on LB + Carb + X-gal plates then they contained a Puc19 plasmid with a kanomycin resistance gene inserted in the MCS. The percentage of transformed cells was also not very high: 0.063%. A way to improve this would be to maybe increase the molarity of the CaCl solution to make the cells more competent. Direct selection method According to the direct method of selection there were no cells that were transformed. This is contradictory to the results obtained from the indirect method of selection. This error could have been produced because of either improper spread plating of plates or because of improper transformation procedure. Also the conditions in the LB + carb + kan plates could have been too harsh(two antibiotics) for the bacteria to pick up growth even if they were resistant to both antibiotics. In following experiments it is better to use the indirect selection method since it seems more successful in selecting desired strains. Comparison of direct VS indirect selection methods The direct and indirect selection methods have both advantages as well as disadvantages. The main disadvantage of indirect selection is that it takes longer since it contains two steps and each step takes at least a day for completion. The main advantage is that if done correctly, the indirect selection methods gives very accurate selection for the desired cells. The reason for that is that first it selects for colonies that just have an insertion in the MCS and this tells the researcher that some type of cloning has occurred in plasmids. The second step then selects for the colonies that contain pUC18 with the kanomycin resistance gene inserted in the MCS. Thus the criterion of indirect selection is that cells have both pUC18 with an inserted DNA in MCS and also have kanomycin resistance. The colonies that grow in the second step fulfill both the criteria. The main advantage of the direct method is that it takes a shorter time to complete and it also uses up less equipment which can also save researchers some money. The main disadvantage with this selection is that it has a higher chance of giving false positives. Direct selection does not select for strains that have DNA inserted in the MCS of Puc18 but only selects for strains that have ampicillin and kanomycin resistance. Therefore the strains that grow in LB + carb + kan plates might have both pUC18 and pKAN plasmids but not the kanomycin resistance gene inserted in the pUC18 MCS. Those strains would still be able to grow since they still have both ampicillin and kanomycin resistance. However the genes would on different plasmids and not on the engineered one. Therefore even though the indirect selection method is longer it is more accurate in selecting the desired strains for this experiment. In conclusion, according to the indirect selection the desired plasmid was engineered by digesting both pUC18 and pKAN with HindIII and BamHI. Also when selecting for cells transformed with pUC18 it is better to employ the indirect method of selection because it gives more accurate results. Question 1: Although both lanes contain plasmid DNA, why doesnt the DNA appear to be in the same location in both lanes? The DNA does not appear in the same location in both lanes because pUC18 and pKAN are of different sizes. pUC18 is 2686 base pais long whereas pKAN is 4194 base pairs long.(Hausner and de Jong 2010) Because pUC18 is of smaller size it will travel farther from the wells than pKAN. Question 2: How would you verify that the transformed cells actually contain the carb/kan plasmid that was used for transformation? One accurate way would be to isolate the plasmid DNA from the transformad cells and run it on an agarose gel. If the kanomycin resistance gene was inserted into pUC18 then on the gel one will be able to see a band of the size 4548 base pairs which is different from both the pUC18 and the pKAN plasmids. The size of the created plasmid was calculated the following way by obtaining the information from (Hausner and de Jong 2010): To find the size of kanomycin resistance gene inserted in pUC18, the number of base pairs from the origin or replication of HindIII was subtracted to the number of base pairs from the origin of replication of BamHI. This was done because pKAN was digested with HindIII and BamHI to obtain the kanomycin resistance gene: 2095 233 = 1862 base pairs The size of the insert was then added to the size of Puc18: 2686 + 1862 = 4548 base pairs