Romeos Tragic Flaw essays

Romeos Tragic Flaw essays In the play Romeo and Juliet, Romeo is the tragic hero who through his tragic flaws, ended the life of his bride and his own. In Shakespeare plays, tragedy is identified as a story that ends unhappily due to the fall of the protagonist. Romeos act of falling in love too quickly and deeply, pride, and poor decision making are his tragic flaws and contributes to the death of Juliet and himself. By analyzing Romeos tragic flaws, it is evident that the play is classified as a tragedy. One of Romeos tragic flaws, being that he falls in love too quickly and too deeply brings him to an awful end. In the beginning of the play we are introduced to Romeo being deeply and hopelessly in love with Rosaline. However, Rosaline did not feel the same way about Romeo. Romeo shows he is unhappy with Rosalines actions when he says to Benvolio: Not having that which makes having short. This very well illustrates how deeply Romeo was in love with Rosaline. In the next scene we realize that Romeo has fallen for another, which is Juliet. We can quickly identify Romeos flaw of falling in love too quickly and deeply when he forgets about his feelings for Rosaline and concentrates on Juliet. Juliet too realizes that Romeos love for her was too fast when she said: It is too rash, too undivisd, too sudden. Romeos strong love for Juliet made him act without thinking when he kept contemplating suicide and saying that he cant live without her. He shows he his love for her when he sees she her dead, not knowing it was a plan, then drinking the poison right away. He shows he will do anything for his new love, including risking getting caught by the Capulet guards at the orchard, and killing himself to be with Juliet. This tragic flaw of Romeo was the reason ...

What to Do in Budapest 11 Top Attractions

What to Do in Budapest 11 Top Attractions SAT / ACT Prep Online Guides and Tips One of the rising stars in European tourism, Budapest’s charms and sights are gaining popularity worldwide. Gone are the days when the Hungarian capital was widely thought of as a drab administrative city, filled only with aging Communist-era buildings. These days, Budapest tourism is rapidly growing and savvy visitors know that it's one of Europe’s great cities with diverse architecture, unique cultural experiences, and some of the most inventive nightlife on the continent. Are you wondering what to do in Budapest? Follow this guide to immerse yourself in Budapest’s history, traditions, and culture. By visiting the Budapest attractions listed below, you’ll find yourself relaxing in opulent thermal baths, paying your respects at moving WWII memorials, admiring the largest Parliament in Europe, and drinking a new cocktail in the gardens of a formerly abandoned warehouse. This list of 11 things to do in Budapest is organized into four groups, based on the location of the sites: Castle Hill, City Park, Parliament and Around, and Central Pest. This organization lets you visit sites near each other, instead of spending your trip running from one end of town to the other. Many people choose to stay in downtown Pest because it has the most accommodation options, but Budapest has a great subway system to help you get from one area to another. You can buy individual subway tickets, day passes, or multi-day passes. I'd recommend that you spend at least a half-day to a full day in each area, and, if you choose to stay longer, you’ll find Budapest has enough activities to fill any itinerary. The Top 11 Places to Visit in Budapest Check out this map of the best things to do in Budapest and then read more about each below. This map can also be viewed online. Castle Hill - Red Matthias Church Fisherman’s Bastion Buda Castle City Park - Blue Szà ©chenyi Bath Vajdahunyad Castle Heroes’ Square Parliament and Around - Purple Hungarian Parliament Building Shoes on the Danube Monument Central Pest - Green Hungarian State Opera House Ruin Pubs Great Synagogue Castle Hill Attractions You can find some of the most famous- and stunning- things to see in Budapest in the Castle Hill area, including gorgeous architecture and historically important sites. Matthias Church Matthias Church is one of the top Budapest attractions, and it’s a center point of Castle Hill. Built in the late 13th century, Matthias Church was the first parish church in Budapest, and it has remained an important symbol of the city. During its long history, Matthias Church hosted multiple royal coronations, was damaged by cannon fire, and spent over 100 years as a Mosque while the city was under Ottoman rule. Even if you feel like you’ve seen enough European churches to last a lifetime, stop for a look at Matthias Church. It doesn’t have the soaring grandeur of larger churches like Notre Dame and Milan’s Duomo, but its unique colors and design make it one of the most pleasant churches in Europe to view. The church was extensively restored in the 1900s, and today it’s popular with tourists who come to see and photograph the Gothic architecture, stained glass windows, and vibrant diamond-patterned roof tiles. The church is also home to the Ecclesiastical Art Museum. The museum (which charges a small fee) begins in the church’s medieval crypt and continues to the chapel. While touring the museum, visitors can view the tombs of 10th century rulers King Bà ©la III and his wife Agnes, replicas of the royal Hungarian jewels, and assorted other jewels and religious objects. Fisherman’s Bastion One of the most unusual sights in Budapest, Fisherman’s Bastion is an elegant, neo-Gothic terrace overlooking the Danube. The site was named for a group of fisherman who defended the city from invasion during the Middle Ages. Walking under gleaming white arches and along staircases and terraces, visitors get sweeping views of the Danube, including a birds-eye view of Parliament. The seven turrets of Fisherman’s Bastion represent the seven Magyar tribe leaders who are regarded as the founders of modern-day Hungary. It may not seem like there’s much to do at Fisherman’s Bastion since the site is little more than an attractive lookout, but many tourists spend far longer than they planned here, strolling from one end of the terrace to the other and taking rolls of photos. When visiting Castle Hill, make Fisherman’s Bastion your first stop (easy to do since it’s very close to public transportation stops). The weather in Budapest can be fickle, and there are times when the terrace and its views are enveloped by fog. It usually clears later in the day, so check back periodically if the weather isn’t cooperating when you first stop by. Fisherman’s Bastion is located next to Matthias Church, and together these two sites make up one the best places to visit in Budapest. Here you can walk along cobblestone streets, look at historic buildings, and grab lunch in one of the numerous traditional cafà ©s that line the streets. Buda Castle Rising up from the southern end of Castle Hill is the imposing, sprawling structure of Buda Castle. The first castle on this site was completed in 1265, and the castle continued to be expanded and rebuilt throughout the centuries. Buda Castle has been a royal residence for generations of Hungarian rulers, besieged by Ottoman armies, taken over by the Habsburgs, occupied by Nazis during WWII, and designated as a World Heritage Site in 1987. Today, the grounds are adorned with numerous statues and gardens, which visitors can admire while walking around the castle. Within the castle are a trio of museums and institutions: the Budapest History Museum, the Hungarian National Gallery, and the National Szà ©chà ©nyi Library (the museum and gallery both require tickets, but the library is free to visit). Each site is well worth a visit, focusing on the history of the city, Hungarian art, and a collection of rare books and paper, respectively. If you can only visit one museum (visiting all three can make for a long day), my pick is the Budapest History Museum. Far more than a dry collection of artifacts, this museum spans three floors and includes such varied exhibits as recreations of opulent rooms as they looked when used by royals, paintings by Hungarian Masters, and excavated rooms of the medieval castle in the basement. There are no cars allowed on Castle Hill unless you live or work in the area, and the lack of noise and traffic contributes to the feeling that you’ve stepped into the past and are far removed from the bustle of the city down below. City Park Attractions City Park is a large public park that includes some of Budapest's best attractions. Szà ©chenyi Bath Visiting the city's famed baths is one of the top things to do in Budapest. Budapest’s abundant thermal waters means there are numerous baths to choose from, but, in terms of history and enjoyability, Szà ©chenyi Bath is far and away your best option. As the largest thermal bath in Europe, Szà ©chenyi Bath is one of Budapest’s iconic sites. When it was built in 1913, the architecture followed the Neo-baroque style popular at the time, and it’s easy to feel like you’re wading through the private pool of a royal palace as you move between the bath’s 18 different pools. When you arrive, you’ll be given a waterproof key card to a personal closet-sized locker, where you can change into your swimsuit (required) and store your belongings while you’re in the baths. There are both indoor and outdoor pools to choose between in Szà ©chenyi. While the indoor pools have a wider variety of temperatures, as well as saunas and steam rooms, most people prefer the outdoor baths because they are typically brighter and cleaner, and they let you enjoy the cheery, slightly aged exterior of the baths. Szà ©chenyi is popular with tourists, and there will likely be a number of people soaking alongside you, but it remains an institution for Budapest locals. They come here to relax after work, socialize, and play a game of chess on one of the waterproof boards in the baths. At Szà ©chenyi, you can also purchase additional services, such as massages. Ticket prices vary depending on the time of day you visit and if you want a cabin or locker, but expect to pay about 5000 Florin (about $18 USD). Massages are an additional 5000-35000 Florin. The thermal waters keep temperatures warm year-round at Szà ©chenyi, so both the indoor and outdoor baths remain open in winter. Vajdahunyad Castle Vajdahunyad Castle is located close to the Szà ©chenyi Baths within City Park, but many people breeze past this tucked away corner of town without pausing to explore. Vajdahunyad can’t compare to Buda Castle’s immense size or history, but this attractive and somewhat mysterious castle earns major points for charm. The castle was originally built in 1896 as a temporary structure of cardboard and wood to celebrate the 1000th anniversary of the Hungarian Conquest of the Carpathian Basin. However, Budapest’s citizens were so taken with it that the present structure, made of stone and painted in muted blues, purples, and oranges, was soon constructed. Set back amongst trees and gardens, the castle’s shadowy exterior reveals features from numerous architectural styles that showcase the variety and evolution of architecture in Budapest. Additional touches to the castle include sunken windows, a portcullis, and numerous turrets overlooking a boating lake turned ice skating rink in the winter. A popular spot for photos is the brooding, slightly creepy statue of Anonymus, a mysterious Hungarian notary and chronicler who lived around 1200 AD. Inside the castle is Museum of Hungarian Agriculture (small fee charged), but even if you’re not interested in the museum’s focus, it’s pleasant simply to walk through the castle’s interior and climb to the top of the two tallest towers for views of the park. Vajdahunyad Castle was only built at the turn of the 20th century, but its historical architecture and romantic setting make it feel far older. A short stroll around the castle or a picnic on its grounds beside the lake is an excellent way to take a break from a busy day of sightseeing. Heroes’ Square Another attraction near City Park, Heroes’ Square does not take long to visit, but it’s one of the city’s most important landmarks. Located at the end of Andrssy Avenue, Heroes’ Square is well-worth visiting to get a sense of Hungary’s history. Built in 1896 to celebrate Hungary’s 1000th anniversary, Heroes’ Square is the largest square in Budapest. The focal point of the square is tall column adorned with a statue of the archangel Gabriel. Flanking the base are seven statues representing the seven original Magyar chieftains. Surrounding the column are 14 pillars with statues of other important Hungarian figures. Viewing the square’s statues and plaques is a great crash-course on the history of the country. Among the statues you’ll see famed Hungarian saints, rulers, and warriors. A plaque in the square dedicates the site "To the memory of the heroes who gave their lives for the freedom of our people and our national independence." During Budapest’s long history, the city was occupied by foreign invaders several times and ruled by a communist regime. Heroes’ Square now serves as a physical monument to Hungary’s dedication to democracy. The square can be visited both during the day and at night, when it is lit up by floodlights. Parliament and Around If you're interested in more recent history, this is the area for you, with monuments to Hungary's tumultuous 20th century. Hungarian Parliament Building The Hungarian Parliament Building is the seat of the National Assembly of Hungary and one of the icons of Budapest. Its design was based on the UK Parliament Building, the Palace of Westminster, but the Parliament of Budapest is no mere copycat. This is one of the oldest and largest legislative buildings in Europe, and since the day it was completed in 1904, it has been the largest building in Hungary. Built along the bank of the Danube River, this soaring Gothic Revival building looks surprisingly delicate up close, with numerous intricate details. Visitors can opt to take a guided tour of the building to see sights including the House Chambers, the inside of the dome, the grand entrance staircase, the Hungarian crown jewels, and a small museum. Tickets, which are 5200 Florin for non-EU citizens, must be purchased beforehand, either online or in person. Make sure you view the Parliament Building both during the day and at night, when the entire structure is lit up, its reflection glowing in the river. Shoes on the Danube Bank Memorial There are World War II memorials far grander than this collection of 60 pairs of iron shoes scattered along the edge of the Danube, but that doesn’t make this site any less moving. The monument was conceived by director Can Togay and created by sculptor Gyula Pauer to honor the people murdered by soldiers of the Arrow Cross, a national socialist party in Hungary during WWII. Between 1944 and 1945, an unknown number of victims, mostly Budapest Jews, were taken to the edge of the Danube and shot, their bodies falling into the river. Before they were killed, they were told to remove their shoes, which could be resold for a profit. This small memorial, a few hundred feet from Parliament, poignantly pays tribute to the victims. The shoes were designed to accurately represent shoes from that period, and they were created in many different styles to represent the wide variety of victims: men, women, business people, teenagers, and children. The shoes are often adorned with ribbons or flowers left as tokens of remembrance by visitors. Walking along the memorial and viewing the empty shoes strewn forlornly along the Danube’s bank is a powerful reminder of the lives lost in WWII. Central Pest Attractions Central Pest is where many visitors choose to stay, because it offers easy access to all the Budapest sights. It's also home to some of the city's most exciting cultural attractions. Hungarian State Opera House Housed in an elegant neo-Renaissance building dating from 1884, the Hungarian State Opera House is a point of pride for many Hungarians, yet it’s much less well-known than many other European opera houses. For a small fee, you can take one of the regular tours of the opera house. After passing the figures of two of Hungary’s most renowned composers in the entrance, you’ll enter a foyer with marble columns and vaulted ceilings. Like all grand opera houses, this one has a sweeping staircase dominating the entrance, used by Hungarian’s past elite to see and be seen. Other opulent touches include a brass chandelier and vast murals decorating the walls and ceilings. The theater itself is decorated with red velvet and gold leaf. It seats over 2,600, with the royal box in the center of the horseshoe-shaped rows. Many people simply opt for a tour, but the Hungarian State Opera House is one of the best places to view an opera in Europe, particularly if you’ve never been to one before. The opera house often performs crowd pleasers like Carmen and Madama Butterfly, which are a great introduction for opera novices. Additionally, tickets here are significantly cheaper than at most other opera houses; you can often get a seat for less than $10 USD. At many other opera houses, tickets this price are typically for the standing area only, and standing shoulder to shoulder with other guests for several hours is enough to fatigue even opera aficionados. At the Hungarian Opera House, you’ll be able to enjoy the opera in comfort without breaking the bank. In addition to operas, the building also hosts ballets and other performances. The Hungarian State Opera House website has a calendar showing all upcoming events. Operas are subtitled in both Hungarian and English, and opera season lasts from September to July. Ruin Pubs Budapest can be heavy with history, so for a change of pace and to get a sense of how the city enjoys itself, head to one of Budapest’s many ruin pubs, drinking establishments that have sprung up in recently un-abandoned buildings. Rumor has it that ruin pubs first came into existence in 2001, out of a need for a cheap place to drink. No two ruin pubs are alike, but all of them share several characteristics: they’re housed in a formerly abandoned building, the furniture is eclectic and often pre-used, and they show their links to the community by hosting local bands or DJs or having local artists decorate the interior. Most of the ruin pubs are located in Budapest’s old Jewish Quarter, in the streets behind the Great Synagogue. In a ruin pub, you could find yourself in a cozy former home with couches for seating, taking to the dance floor in a large, rambling warehouse, tasting plinka (a traditional fruit brandy) under the glow of old-fashioned lanterns, enjoying beers in a courtyard filled with picnic tables, or learning Hungarian phrases at one of the regular informal classes the pubs host. One of the best aspects of ruin pubs is that you never know what type of experience you’re going to get until you walk inside. The best ruin pub to start at is the original, Szimpla Kert. Occupying a large, formerly abandoned building, Szimpla is filled with mismatched and often antique furniture and decorations. Outside there’s a spacious courtyard which includes an old Trabant car guests can lounge in. Ruin pubs attract a mostly young, international crowd, but you’ll find all types here. Local Hungarians often frequent them, as do older visitors. Ruin pubs may be most enjoyable to visit on a warm summer evening, but they’re open year-round, with outdoor spaces typically closed in the winter. Great Synagogue This massive building, also known as the Dohny Street Synagogue, is the largest synagogue in Europe and the second-largest in the world. It was completed in 1859 in the Neo-Moorish style, giving it a distinct look compared to many other synagogues. Inside the Great Synagogue is seating for more than 3,000 people, as well as the Jewish Museum, which has exhibits on Jewish traditions and the history of Judaism in Budapest. The building is rich with history. During World War II, the site was bombed by Nazi planes, and it later became one border of Budapest’s Jewish ghetto. In the grounds surrounding the synagogue is a graveyard where over 2,000 people who died in the Jewish ghetto during the winter of 1944-1945 are buried. There's also the Heroes’ Temple honoring Hungarian Jews who died during World War I and the Holocaust Memorial. The Holocaust Memorial, also known as the Emanuel Tree, is particularly striking to see. Crafted by Hungarian sculptor Imre Varga, the memorial takes the form of a delicate and intricate weeping willow. The name of a Hungarian Jew killed during the Holocaust has been engraved on each metal leaf. In my opinion, this is one of the world’s most beautiful and moving Holocaust memorials. If you don’t have time to tour the synagogue, you can get a view of the tree through the gate surrounding the grounds. You can take a self-guided tour around the synagogue, but a guided tour (available at the entrance) can help you navigate the large site and fully appreciate its historical importance. Entrance to the synagogue costs 3000 Florin and includes access to both the building and its grounds.

The intersection between ethics and politics in ANIMAL RIGHTS about Essay

The intersection between ethics and politics in ANIMAL RIGHTS about Animal Genetic Testing - Essay Example Animal testing is one of the most ethically gray points in society. One can say that it can is a necessary evil as it is an important factor in keeping technology, as in medical technology. We need to know how certain chemicals can work in our body when dispensed, hence animal testing. In short, the development of drugs is one of the reasons why there is animal testing in the midst. However, we are also aware of the fact that animal testing is brutal as it is (most of the time) done without painkillers – meaning, the animals would suffer for the plight of the people, plight of the people that make them miserable and take their services for granted, as they are inferior – animals as they are called. There are about 20 million animals that are killed every year for experiments. 75% of the said figure are killed because of medical testing and that the rest of them are killed to try new products (as in animal testing). Ten percent of the 20 million figure are said to be und ergoing procedure without any pain killers. There are many people, especially the animal rights advocates, who oppose the use of animals in medicine and health services, that is true. They are imposing various government to give restrictions on companies and businesses that do not respect animal rights for research. However, this movement is also taking a call to stop as there are also a growing number of people who do not want to stop experimenting on the animals. This is due to the fact - or fear – that medical research and technology would stop growing at the rate that it is growing now. This means that the stoppage of the use of animals in research would curtail the growth of the knowledge and scientific progress. Pain is an intrinsic evil. Those people who argue that painful experimentation on animals should be avoided, or stopped (in a perfect world), say that, and anything (or anyone) that causes pain to another creature or animal is immoral. Using the words of Jeremy Bentham, one cannot say (or use the argument) that the animals â€Å"can’t talk, or reason† hence they can be used. The real question that should be asked is â€Å"do animals suffer?† The answer is yes. They do suffer because they feel pain. The scientist who conducts research the rat for some new formulation about a new drug knows that the rat feels pain because it needs to know how the human would react if it is subjected to the new drug – will the new drug cause pain to the patient or not? Every writhe and struggle of the anilam is recorded and then will be used to improve and design a new drug. Even the fact that the scientist will have to let the animal choose to what its setup is: to have electric shocks or to have hunger, is immoral. Although the data will be used and collected to know whether ulcers of some kind would be formed, the doctor still uses the animal to know how the human mind works as the rat and humans have the same neural responses. Basically, we are like rats. Pain is an intrinsic evil. It does not matter if it is experienced by a man, child or animal. If it is wrong to inflict pain on a human being, then it is wrong to inflict pain on an animal, or any creature for that matter. Another thing that should be

Lesson Plan Reflections Essay Example | Topics and Well Written Essays - 500 words

Lesson Plan Reflections - Essay Example The activities I planned supported the learning objective since they were related to the lesson work and aimed at enabling the student understand the concepts for every lesson. For example, reading the story on time and pointing to the clock to depict the different time mentioned engaged the students since it entailed use of both audio and visual learning aides. This fostered the students to meet the learning objectives. The other activity for the math lesson on time around the world required the students to draw picture to indicate digital times as well as analog time. This meets the objective, which was to enable students to tell time using analog and digital clocks. I could tell whether the students were successful in achieving the objectives depending on how they performed in the after lesson assessments. For example, the objective of the Social Studies lesson on Girl on the Go was to enable students remember the different countries they had learnt in the history lesson. The story used different monuments in association with different countries. To assess if the students met the objectives, I asked post-reading questions that covered what happened in the beginning middle and end of the story. Moreover, I asked the students to mention the countries that Isabella visited, what the different countries were famous for, and they answered correctly. Additionally, the students were supposed to tell each other countries they had visited, to see if they remembered the previous lessons. I realized that student learn better through activities. For example, after reading the story about the Girl on the Go, I realized that students could easily remember the courtiers mentioned in the story as compared to other countries covered in previous lessons. They would also associate different monuments with the countries where they are located. Moreover, use of pictures and a clock in the math class made the lesson interesting and the students were excited when they realized

Expression and Purification of recombinant Green Fluorescent Protein

Expression and Purification of recombinant Green Fluorescent Protein ABSTRACT: The purpose of this experiment was to determine if a His-6 tagged recombinant form of Green Fluorescent Protein could be expressed in a pRSETA vector of E. Coli. This was determined through multiple procedures beginning with purifying the sample with Ni +2 agarose chromatography which showcased the relative fluorescent activity of the samples, which elution sample two (E2) had approximately 100,592.2 RFU/mg . The yield of total protein was found by use of a Bradford Assay and a standard curve. The purity of the GFP was determined by comparing the intensity of bands that appeared at around 31.4 kDa (the molecular weight of rGFP) to a molecular weight ladder on an SDS-PAGE gel. The Western Blot test, utilizing a nitrocellulous membrane, confirmed the expression of rGFP. The Western Blot confirmed that the correct bands were analyzed in the SDS-PAGE gel which E3 had an estimated purity of 0.4, indicating a yield of approximately 29.4 ug of rGFP for the third Elution (E3) after a total p rotein amount of 49 ug was extrapolated from the Bradford assay. INTRODUCTION: The Green Fluorescent Protein was first isolated from the Aequorea Victoria jellyfish and studied by Osamu Shimomura. In other organisms other than this specific jellyfish, there are fluorescent proteins with more color varieties than just green (Ward). GFP is able to fluoresce due to the formation of a chromophore in its center that resides in a stable beta barrel structure. The interactions between Ser64 and Gly67 on an alpha strand form a five member ring structure and its this structure that contains properties for fluorescence. When ultraviolet light is absorbed at 395nm, an emission wavelength of 510nm is seen as fluorescent green light. The excitation wavelength of 395nm excites the electrons within the protein and raises the energy of the protein. As the protein loses energy, it emits the energy at a wavelength of 510nm. Wild type GFP does not have immensely visible fluorescence, therefore a genetically modified form of GFP, GFPuv, was used in order to better observe fluoresc ent activity. For rGFP to be fully expressed, GFPuvs open reading frame was pRSETA vector, which was cut by a restriction enzyme at sites that complimented the start and stop codons of the His-6 tag sequence. A T7 promoter and Xpress Epitope were also genetically added into the vector with the purpose of simplifying purification. T7 polymerase minds to the T7 promoter and activates the production of GFP, while the Xpress Epitope allows for the binding of a primary antibody. The binding of a primary antibody allows for the binding of a secondary antibody which contains horseradish peroxide, producing bands on the nitrocellulose membrane. (2 pg 351) Histidine-6 tags primary purpose is to aid in the purification of rGFP proteins in affinity chromatography. Histidine has an affinity to bind with transition metal ions, and is why a Nickel+2 agarose chromatography was chosen. When a sample is passed through the Nickel+2 agarose column, the His-6 tags fused with rGFP protein will bind to the column and any proteins not of interest or containments will pass through the column. To elute the bound rGFP proteins from the column, elution buffer containing Imidazole is used and competes with the histidine residues for the binding sties to the Nickel+2 agarose column due to its higher affinity for them. Elutions from the column therefore contain rGFP. (2 pg 359) The purpose of this experiment was to see if a His-6 tagged recombinant form of GFP, from the E. coli strain BL21pLysS, could be expressed and then purified using Ni+2 agarose affinity chromatography technology. If this experiment is successful, an inexpensive and relatively simple method to measure gene expression and locate and track proteins may be at hand. MATERIALS AND METHODS: Bacterial Expression Grow two bacterial cultures: G, containing BL21, DE3, pLysS, and the plasmid pRSETA-GFPuv with the GFP sequence, and V, which is identical to G, but it does not have the GFP sequence. Incubate both cultures at about 37Â °C until OD600 equals 0.5. Transfer 1ml of each culture into separate centrifuge tubes and centrifuge to obtain a pellet. The supernatant from the tubes will be discarded and then label the tubes V0 and G0 and stored at -20Â °C. Induce the rest of the culture with IPTG and allow to grow for 3 hours. After the 3 hours, collect 1ml of each culture and centrifuge. Label the tubes V3 and G3 and were store at -20Â °C. Collect 15 ml of G and centrifuge, and label it as G3-15ml and store at -20Â °C. (1 pg 105) Preparation of Crude Extract After the slow freeze process, add 1 ml of breaking buffer (10mM Tris, pH 8.0; 150mM NaCl) to the frozen bacterial pellet G3-15ml, and pipette up and down until completely dissolved. Then transfer the contents to a centrifuge tube and vortex for 5 minutes and place in a 37Â °C water bath for 10 minutes. Afterwards, incubate in dry air at 37Â °C. Centrifuge the mixture at 4Â °C and transfer the supernatant into a clean centrifuge tube and take a small sample to label as GCE. The remaining supernatant will go through the Nickel+2 agarose column. (1 pg 110) Ni+2 NTA Chromatography Pack a 3ml plastic syringe with a small amount of glass wool to prepare the Ni+2 agarose column. After that, pipet 1 ml of breaking buffer into the syringe to remove any air bubbles. As the breaking buffer is going through the syringe, pour some breaking buffer into the leur-lock and then screw it onto the syringe. Add 1ml of 50% Ni+2 agarose into the column and then open the leur-lock to pack the agarose matrix in the colum. Add 5 ml of breaking buffer subsequently into the column to push the ethanol of the Ni+2 agarose through the column. Then close the leur-lock and apply the crude extract to the column and allow 5-10 minutes for the histidine tags on the rGFP to bind with the Ni+2 agarose beads. Open the leur-lock and collect the non-crude extract in a centrifuge tube labeled W1 for the first wash. Add 0.5ml aliquots of breaking buffer to the column and collect the washes in tubes W2-W10. Add 10 increments of 0.5ml of elution buffer and collect in tubes E1-E10. Then store your GC E, W1-10, and E1-10 samples at -20Â °C. (1 pg 111-2) Bradford Assay Analysis To create a Bradford assay, mix protein sample with water, and then add Bradford reagent dye. Vortex the mixture and then transfer it to the wells of a microtiter dish, so that the absorbance of your protein sample can be determined at 595nm using a spectrophotometer. To determine the amount of total protein present in the sample volume, you must create a Bradford standard curve using 0, 2.5, 5, 10, 15, and 20 ug of BSA and the absorbance values observed to determine a best-fit line. Perform the assay in singlicate using samples W1-W6 and E1-E6 to determine if the microplate data falls within the standard curve. Once you have determined what volume of sample to use, repeat the Bradford assays two more times for each sample. Use the standard curve and experimental absorbance values to extrapolate the total protein present in the volume of sample you use. (1 pg 124-6) SDS-PAGE/Coomassie Blue Analysis of rGFP Fractions Create a 12% Resolving Gel by mixing water, 30% Acrylamide, 10% APS, TEMED, and 4x resolving buffer [0.75M Tris pH8.8, 0.4%SDS]. Pour the resolving gel into a gel electrophoresis set up and overlay with some water. Allow the resolving gel to polymerize and proceed to making a 5% Stacking gel by mixing water, 30% Acrylamide, 10% APS, TEMED, and 4x stacking buffer. Pour the stacking gel on top of the resolving gel and immediately insert a comb to form the wells. Prepare your loading samples with 4x sample loading buffer and then vortex, boil, and centrifuge. Do this for the G0, G3, GCE, W2, W3, E2, and E3 samples (or fractions that contained the most rGFP fluorescence). (1pg 111-2) After the gel has solidified, transfer the apparatus into the electrophoresis tank and load the electrophoresis buffer into the tank. Then load your samples into lanes one through seven respectively along with a marking ladder to compare the distances the samples traveled after electrophoresis. Electrophorese at 200V for approximately 45 minutes and then stain your gel with Coomassie Blue dye. To remove the stain that has not been absorbed by proteins, the gel is soaked in dilute acid and methanol. Western Blot Develop an SDS-PAGE gel as previously described using your sample fractions and transfer the proteins onto nitrocellulose transfer membrane using two locking cassette lids with sponges and filter paper encompassing the gel and nitrocellulose. Remove the nitrocellulose and stain the membrane with Ponceau S stain, and allow to incubate for approximately 2 minutes. Once stained, wash the membrane with nanopure water until bands are visible on the membrane. Next, perform the blocking step by placing the membrane in a container containing 5% non-fat dry milk/TBS solution and incubate on a shaking platform. Pour out the blocking solution and add 0.05% Tween 20/TBS solution and then allow it to incubate on a shaking platform, and then pour out the solution. Repeat this wash step two more times. Add mouse IgG anti-Xpress epitope MAb and allow incubate on a shaking platform this is the primary probe step. Then repeat the wash step as mentioned previously three more times, and add Sheep IgG a nti-mouse IgG conjugated horse radish peroxidase polyclonal anti-serum solution (the secondary probe). Allow the membrane to incubate, and then perform the wash step twice. For the final wash, use only TBS, and then add TMB substrate solution and incubate until appropriate banding is shown. Stop the reaction with distilled water and scan or photograph the membrane as the results may fade with time. RESULTS: In the specific strain of E. coli, BL21pLysS, pRSET-GFP represses the lac promoter which is part of the operon needed for T7 polymerase activity. T7 polymerase binds to the T7 promotes of pRSET-GFP and activates the His-6 tag that expresses the fluorescence of GFP. Even though some GFP is produced this way, it is experimentally not enough, so IPTG is added to repress the repressor so an increase of GFP production can occur. V0, V3, G0, and G3 were all bacterial cultures of E. coli with specific distinctions between all of them. V0 and G0 were collected from bacterial cultures that were not yet induced, but G0 contained the GFP sequence while V0 did not. V3 and G3 were the V0 and G0 samples after induction had occurred for 3 hours, and again G0 contained the GFP sequence while V0 did not. W1-W10 were washes collected from the Ni+2 agarose column using the breaking buffer whereas E1-E10 were washes collected using elution buffer. Figure 2. SDS-PAGE/Coomassie Blue stained gel analysis of rGFP SDS-PAGE gel, which was used to analyze our purification of rGFP from a crude extract, and the corresponding molecular weight. This gel was made with a 12% resolving gel and a 5% stacking gel. Wildtype rGFP has a molecular weight for 27kDa. The ladder has kDa rungs of 97.4, 66.2, 45.0, 31, 21.5, and 14.4. The band for rGFP has been indicated on the figure. The different samples were taken during our purification. The highest protein containing washes and elutions were used in the SDS-PAGE gel. G0 represents a sample containing rGFP prior to induction. G3 represents a sample containing rGFP after 3 hours of induction. GCE represents the GFP crude extract after the slow-freeze/quick-thaw process. Figure 3. Western Blot of rGFP We used the Western Blot to detect, and more importantly, quantify proteins that react with antibodies. The Ponceau S stain was performed to highlight the existence of our proteins successfully being transferred from the SDS-PAGE gel. We then performed a blocking step with 5% non-fat dry milk/TBS solution followed by washes with 0.05% Tween20/TBS. The probe first used was mouse IgG anti-Xpress epitope MAb solution, which we followed with washes of Tween20/TBS solution. The second probe used was sheep IgG anti-mouse IgG conjugated horseradish peroxidase polyclonal anti-serum solution, which we followed with two washes of Tween 20/TBS solution with the third wash being pure TBS. Then, TMB solution was added to develop the nitrocellulose membrane and this reaction was stopped by pouring distilled water over the membrane. Above are the 8 lanes, G0, G3, GCE, W2, W3, E3, E4, and a known molecular weight ladder, respectively. CONCLUSION/DISCUSSION: A great amount of research has been put into the field of monitoring gene expression and there are currently many research laboratories that use labeled antibodies and other various means to do so. However, this new recombination technology, a vector can be created to provide a simpler and less expensive approach that can even be performed in vivo. The purpose of this experiment was fulfilled as His-6 tagged rGFP from a particular E. coli strain was successfully expressed, purified, qualified, and quantified by means of Ni+2 agarose chromatography. GFP was initially expressed as a histidine tagged protein that was inserted in E. coli and subsequently a crude extract of this protein was isolated. Throughout the experiment, we were able to inspect and measure the fluorescing activity of our elutions with the use of handheld UV lights. GFP was successfully expressed because of the fluorescent activity that was emitted when shown under UV light. This rGFP was purified using a Ni+2 agarose column which allowed for the binding of the His-6 tagged rGFP to the Ni+2 agarose beads in the column, and subsequently let all other proteins not of interest and containments to be collected in washes. Then, rGFP proteins were eluted by passing elution buffer containing Imidazole through the column because Imidazole has a higher affinity for the Ni+2 agarose beads than does Histidine. Therefore, one could expect that most if not all of the rGFP would be found in the elution washes and would present higher fluorescing activity than the washes with breaking buffer. Figure 1 clearly supports this argument. From referencing Figure 1, the most rGFP was found in E2 because it exhibited the highest fluorescent activity in RFUs. Using a standard curve based off of known quantities of mass of a certain protein, one is able to extrapolate the amount of protein present in ones sample. This can be used to estimate the amount of rGFP activity. For instance, E2s specific activity was found to be approximately 100,592.2 RFU/mg. W1-W6 had the least amount of fluorescing activity because the washes were done prior to the addition of Imidazole through the column. This is expected as Imidazole would flush out rGFP in the elutions, which would produce the highest GFP fluorescing activity. Percent purity of the rGFP samples were approximated using the SDS-PAGE gel with Coomassie blue staining to allow visible bands to appear which were used for comparisons. The molecular mass from each individual sample was determined by comparing the bands with the bands of the known weight ladder. The percent purity of each individual band was determined by comparing the bands against each other. The molecular weight of wild type rGFP is 27 kDa, and the molecular weight of rGFP for mutant rGFP used for this experiment was approximately 31.4 kDa with the 40 extra amino acids that were added taken into account. For example, E3 percentage of purity was found to be .4. The amount of protein yielded was 29.4 ug of rGFP for the third Elution fraction (E3) after a total protein amount of 49 ug The Western Blotting technique detected and quantified proteins that reacted with a specific antibody. In our case, it verified rGFP was expressed and whether or not it fell in the range of the expected molecular weight of 31.51 kDa. Lanes GCE, G0, G3, and E3 all contained a bright band around 31.4 kDa, indicating that rGFP was existent. This result corresponds to the expected of E3 having the one of the highest rGFP activity. The fact I had used E3 for the SDS-PAGE and Western Blotting skewed my results, but E2 and E3 had the highest activity for me. Even with my error, GFP was effectively expressed and purified and its finding is important to science because it can be used to track cancer causing cells one day to help figure out where problem spots begin and can help eliminate the issue before it escalates indicating that fluorescent proteins might eventually be clinically useful in cancer patients (Hoffman). This can occur since GFP can successfully be expressed in other organisms as this experiment through the use of E.coli, humans cells can certainly be tagged and label the tumour before treatment and then monitor for fluorescence after treatment to identify possible recurrence or metastases (Hoffman). The same can be said with heart disease and other health related problems. The future of GFP remains limitless when scientists can unlock its full medical potential. REFERENCE/BIBLIOGRAPHY (1) Rippel, Scott. BIOL 3380 Fall 2010: Biochemistry Lab Manual. The University of Texas at Dallas. Richardson, TX. (2) Rippel, Scott. BIOL 3380 Fall 2010: Biochemistry Lab Lecture Notes. The University of Texas at Dallas. Richardson, TX. (3) Ward, William. History of GFP and GFP Antibodies. Brighter Ideas. 2009. 26 Oct, 2010. http://www.brighterideasinc.com/proteins-antibodies/history-of-gfp-and-gfp-antibodies/ (4) Ninfa, Alexander J. and David P. Ballou (1998). Fundamental Laboratory Approaches for Biochemistry and Biotechnology. (5) Hoffman, Robert M. Uses of Fluorescent Proteins to Visualize Cancer In Vivo: Can Fluorescent Proteins be Used in Humans? 2005. 28 Oct. 2010. http://www.medscape.com/viewarticle/513975_6

Library system Essay

Chapter I Introduction The world of Information Technology transformed and made the life of human beings easier through the innovation of different machines and software applications. Faster and reliable sources have been offered by search engines which are all over the web. But even though this is the case, many schools and universities are still requiring their students to use library materials in their researches and school works. From the statement of purpose, the compute’rized library system: meeting information needs of the people of Saint Michael College of Caraga, Nasipit, Agusan del Norte. Our proposed system, the Saint Michael College of Caraga Library Management System, is a system wherein there is no need of manual library transactions. This will carry out different processes such as searching books, keep records of the books, borrowing of books and return of books. Hence, at present, Saint Michael College of Caraga is maintaining the School Library in a manual basis. Handling bulky records in a manual method is very difficult. And the process of updating could not be done easily and  accurately. Likewise, a manual procedure of handling bulky record is very slow and is prone to manual errors. Consequently, it is proposed that Saint Michael College of Caraga shall maintain an electronic data base management system (DBMS) for the purpose of maintaining the records of the School Library for easy, fast and accurate processing and maintenance of these records that will enhance management decisions and for the improvement of its services to all the stakeholders of Saint Michael College of Caraga. Library is regarded as the brain of any institute; many institutes understand the importance of the library to the growth of the institute and their esteem users (students). Library Management System of Saint Michael College of Caraga supports the general requirement of the library like acquisition, cataloguing, circulation that offers many flexible and convenient features, allowing librarians and library users to maximize time and efficiency. Library System gives the all detailed information about students, staff and books. It will track on the how many books available in library and books issued to the students. It shows popular book among the students. It will provide book lost in library. It keeps the record of the suppliers and book binders. It generates MIS reports for management. Our software is customizable for any library requirement. In this chapter we take opportunity to consider changes in the library services, not in terms of minor, short term change in equilibrium of the sort just noted above, but in longer term. There has been continuing concern about the future of the libraries. Given the extent to which the provision used and the libraries are influenced by the social environment, and given possibilities by the use of new information technology; it would be unreasonable to expect libraries to remain static. But if not, what would be the nature of the change? The issue is not whether there is a change but what will be the change. If we are to make a claim to understand the nature of library services, the surely we ought to have notions, some forecasts, about how library system might change. Purpose and Description In setting up a library, one aspect that should be considered, what are the resources the library has and what are the ways to provide a better service to the students? Well, one of the easiest ways is having a system to  organize all the transactions in the library. A library system is software that will handle basic and systematic organization of function in the library. The system would provide basic set of features to add/update student’s information, add/update books information, search for books and manage check-in/check-out processes. In this application we can maintain the records of students and books and enable to determine how many books are issued and likewise determine the available books in the library. The proposed library system will greatly improve the efficiency of the school library. This study has the following hypothesis: The profiles of the respondents taken are their names, Student ID Number, Year and Course, and Title and Author of the Book Borrowed. The manual system is very time consuming, inconvenient when it comes to recording, organizing and retrieving borrower’s record in the log book. There are few solutions but many a times, we â€Å"making them works† by finding â€Å"ways to work around† system inadequacies. Moreover we have limited staff resources, these â€Å"work around† waste time, effort, and skills that should be spent on user services. solutions do not keep up with the technological changes and hence prove to be time wasters. Library Management system is a small footprint software suitable for personal /individual Libraries. You can store the information about the books and other material and control the movement of the same. Silent Features †¢ Control the movement of books and other material and avoid losing the same. †¢ Search if you have a specific book in your collection based on t he title, author etc. †¢ Print the spine labels for the book. †¢ Find what a specific person has borrowed from you. Objectives The main objective of the application is to automate the existing system of manually maintain the records of the Book Issue, Book Return from the student, Stock Maintenance, and Book Search to be computerized. And to develop a database which stores user details and book details, give reliable search facility for the user, create an easy to understand user friendly environment. So the Book Issue, Return, Searching will be faster. This  application can be used by any Library to automate the process of manually maintaining the records related to the subject of maintaining the stock and Book Issues. The general objective of this study is to design and develop a library system that will serve as a proposal to help librarians save time with the automation of its daily operation. Specific Objectives: To computerize records keeping of books; To allow librarians to retrieve complete information of the book and its borrowers. To check the availability of the books and penalties. To search, issue and return of books. It can only be utilized by the librarian Scope and Limitation The study only focuses on the Information Technology Library Management System of Saint Michael College of Caraga. Without computers, as some libraries are, all of them are dependent on paper work. When compared to computerized systems, data backup and data retrieval systems are inefficient and labor intensive. Users of a paper based system become almost wholly reliant on the librarians, for all levels of service, whether they are enquiries about existing books, inter-library loans, or the availability of books. There is additionally no real involvement of a user in the entire process. The transactions that the system accommodates are the attendance of every student that enter the library, book registration and deletion, updating book information and searching thesis references, Borrowing of books, and keeping the record of transaction. Review of Related Literature This chapter contains the relevant outline of literatures related to the study of Library system. This study composed of programming languages, Database and Graphical User Interface (GUI) that is used in the system. This system would be used by members who may be students or professors of that University to check the availability of the books and borrow the books using automated device, and by the librarian to update the databases. The purpose of this document is to analyze and elaborate on the high-level needs and  features of the Library Management System. It focuses on the capabilities and facilities provided by a Library. The details of what all are the needs of the Library Management System and if it fulfils these needs are detailed in the use-case and supplementary specifications. Automated book monitoring system helps to reduce the effect of entering the wrong quantity and the amount of staff time devoted to repetitive activities. Related Studies According to ACRL (1994) suggested that institution should be prepared to utilized new technologies for accessing information as they are developed. This is why operating the library operations is quite a welcome idea. According to Jeff Kaplan (05/08/2007 A new generation of automated network management software and services is helping in-house staff address this challenge. These let network professionals establish regular patch-management procedures to safeguard against escalating security threats; and create system monitoring routines to identify load imbalances, which could cause service disruptions or performance problems. They also discover, inventory and track assets to make sure that hardware and software licenses are up-to-date and that problems can be resolved faster. According to Voustin Sweere (Aug.2001) Nowadays every book keeping system used in practice is automated. Most book keeping software an integrated information system are based on database. In this paper, we develop a conceptual book keeping model which is not based on manual techniques, but which is applicable in database environment. Technical Background In this chapter, it provides an overview of all the overall system design, features and functionalities. The system is design to implement a computerize library management system in Saint Michael College of Caraga. This system consists of one user, the administrator. The administrator can  access the system by having first the login security which they have to input his/her user name and password. Once the inputted username and password did not match, the required/registered information it displays â€Å"Invalid Username and Password†. If the username and password match the registered information, the next form will display depending on the role of the administrator.

The Student Loan Market Is A Growing Component Of The U.s....

The student loan market is a growing component of the U.S. economy. Student loans are the second largest source of household debt. Assisting borrowers with the loan repayment process, like finding the right repayment plan and avoiding default, is an essential component of preventing further drag on the economy. Further, illuminating practices unfriendly to consumers and improving student loan servicing can potentially decrease the default rate and expedite the repayment process. To end these practices, we recommend that the CFPB use its authority to promulgate regulations and implement new student loan servicing protections for borrowers in the following ways: 1. Require student loan servicers to do their part to counsel borrowers about their repayment options using consistent, industry-wide policies 2. Require servicers to send emails to borrowers recapping each interaction or transaction 3. Require student loan servicers to follow borrowers’ payment instructions 4. Issue guidance for student loan servicers to follow when performing account transfers Many borrowers try their best to repay their student loans but encounter unfair policies designed to extend their repayment periods. In April, we conducted an online survey to explore the â€Å"borrower experience† during repayment. We learned that borrowers are doing all or the right things: they borrowed money to get an education necessary to open doors to opportunities. When they entered repayment, however, the frustration,Show MoreRelatedThe Duration Of Unemployment As Provided By The Bureau Of Labor Statistics1704 Words   |  7 Pagessignificantly to 33.7 (BLS 2015). The average hours in 2006 before the Great Recession were also around 34.5 hours, so I would say the labor market has done a good job recovering from the recession (BLS 2015). Even two years ago it seems that we had not fully recovered, when the average weekly hours worked hovered around 34.4 (BLS 2015). 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