Analysing isolation of DNA plasmid and Agragose of gel electophoresis

Analysing isolation of DNA plasmid and Agragose of gel electophoresis Introduction (a) The aim of this experiment was to successfully isolate a DNA plasmid from E.Coli cells (Escherichia coli). We then use commonly performed a method commonly used in biochemistry and molecular biology called agarose gel electrophoresis. This is used to separate DNA and RNA fragments according to length are used to estimate the size and charge of the DNA and RNA fragments or to separate protein by size. In this procedure as stated above, we used e.coli as these are plasmid containing cells. These cells were placed in a buffer and mixed with a solution of 1% (w/v) SDS (sodium dodecyl sulphate) which was mixed with sodium hydroxide. The alkaline solution (12.6PH) causes the molecular weight increases this causes it to become like chromosomal DNA. Using alkaline lyses is based on differential denaturation of chromosomal and plasmid DNA in order to separate the two. The double stranded plasmid and chromosomal DNA is converted to single stranded DNA due to the lyses of the cells which solubilises protein and denatures the DNA. Subsequent neutralization is potassium acetate allows only covalently closed DNA plasmid DNA to reanneal and stay solubilized. Chromosomal and plasmid DNA precipitate in a complex formed with potassium and SDS which is removed by centrifugation. Protein dodecyl sulphate complexes are precipitated die to it being insoluble in water. When centrifugation neutralizes the lysine it yields to a minuscule supernatant fraction that contains plasmid DNA a network of chromosomal DNA and protein Plasmid DNA is concentrated by from the supernatant by ethanol precipitation. Plasmid DNA isolated by alkaline lyses is suitable for most analyses and cloning procedures without further purification however if the isolated plasmid DNA is sequenced and additional purification step such as phenol extraction is used. (b) The aim of Agarose gel electrophoresis is to analyse the plasmid DNA that was extracted from the procedure before. The technique of electrophoresis is based on the fact that DNA is negatively charged at neutral pH due to its phosphate backbone. And like any other biological macromolecules can move within an electrical field. The rate of the DNA slows down when its moves towards opposite poles because of the agarose. The agarose gel is a buffer solution this is used to maintain the required pH and salt concentration. The agarose forms hole or wells in the buffer solution and the DNA inserted in through the holes to move toward the positive pole. As mentioned before the agarose gel slows down the rate of DNA so the smaller DNA moves faster than the larger molecules of DNA as the smaller ones fit through the whole easier. This causes the DNA to be separated by size and can be seen visually. To make the electrophoresis to function and separate DNA molecules it must contain an electro phoresis chamber.and power supply, combs which are placed in the chamber this is how wells are formed when agarose is placed in the gel, Trays that contains a special gel that comes in many sizes and and have UV-properties combs which is how wells are formed when agarose is placed in the gel, Electrophoresis buffer, Loading buffer, which has a thick consistancy (e.g. glycerol) so the DNA can be easily placed in the wells and one or two tracking dyes, these travel in the gel and help visualize how the process is being carried out and to moniter how far electrophoresis undergone. Ethidium bromide, is a dye used to stain the nucleic acids.  . Tran illuminator  (an ultraviolet light box), which is used to visualize ethidium bromide-stained DNA in gels.   Method for plasmid isolation 1.5 ml of culture that contains E.coli cells containing the plasmid pUC118 was inserted into an Eppendorf tube. This was then centrifuged at 13000 rpm for two minutes The liquid contained in the Eppendorf tube was discarded carefully by using a pipette and then inverting the tube on a test tube to remove remaining drops of the liquid without removing the bacterial pellet 200 micro-liters of solution A was added to the bacterial pellet. This ensured that the suspension is homogenized (mixtures are well separated 400 micro-liters of solution B was then added and mixed well these solutions contain the SDS and sodium hydroxide. This neutralizes the solutions 300 micro-liters of solution C which contains the potassium acetate which was also mixed and then was incubated on ice for 10 minutes This mixture was the centrifuged at 13000rpm for 5 minutes 750 micro-liters of this supernatant was transferred to a new Eppendorf tube whilst ensuring none of the precipitate was interfered with 10 micro-liters if RNAse solution was added to a duplicate tube and labeled as R+ 450 micro-liters of isopropanol was added to each test tube and mixed well This was then centrifuged at 13000rpm for 5 minutes The supernatants were then carefully removed and the DNA was retained 400 micro-liters of ethanol was added and allowed to stand for a minute it allow the salts to dissolve the liquid was carefully removed so as not to remove the DNA precipitate. The sample was then allowed to dry at room temperature Each pellet was then dissolved in 10 micro-liters of TE buffer Q1 The viscosity after 400 micro-liters of solution B was added and mixed a low viscosity was observed as it had a very watery texture. Q2 there was no viscosity after the transfer of 750 micro-liters of supernatant to a new eppendorf (a) Agarose gel electrophoresis The sample obtained from the experimental procedure above were then examined using the method of agarose gel electrophoresis The RNAse treated and untreated plasmids were examined. 10 micro-liters of loading buffer was added to 10 micro-liters of DNA for each sample The samples containing DNA mixed with loading buffer were then pipetted into the sample wells, and a current was applied. This was carried out for 30 minutes It was clear that the current was flowing as bubbles were observed to be coming off the electrodes. The negatively charged DNA migrated towards the positive electrode at the distal end, (which is usually colored red) It was analyzed that the smaller DNA molecules travelled quickly through the gel which showed that the procedure was carried out successfully as the DNA was separated according to size Results/ Discussion (a) Isolation of DNA plasmid The DNA plasmid was successfully extracted from the E.coli cells and then the DNA was the successfully separated according to size by using the agarose gel electrophoresis method. Solution A contains 25 mM of Tris-HCL (pH 8.0)50 EDTA. Tris is a buffering agent this maintains a constant pH. The EDTA is used to protect the DNA from DNAses which are degradative enzymes; the EDTA also binds divalent cations that are necessary for DNAse activity. The solution B contains SDS which is a detergent and NaOH. This neutralizes the solution, the alkaline mixture also causes the cells to rupture and the SDS the lipid membrane is broken apart and the cellular proteins are solubilized, NaOH converts the DNA into a single strands which is caused by denaturation. The solution C contains potassium acetate (pH 4.3) the acetic acid neutralizes the pH, allowing the DNA strands to renature. The potassium acetate is added its causes the SDS to precipitate, along with the cellular debris. The  E. coli chromosomal DNA is also precipitated. The plasmid DNA remains in the solution. The viscosity of this is very high as it has a very gel like texture. When the supernatant is placed in a new eppendorf tube after 5 minutes of centrifuge this causes the plasmid DNA to separate from the cellular debris and chromosomal DNA in the pellet. The isopropanol is then added this pulls the plasmid out and causes it to precipitate nucleic acids. After centrifuge a small white pellet was observed at the bottom of the tube after the supernatant was carefully removed this further purifies the plasmid DNA from contaminants. 400microliters of ethanol was added this washed the residual salt and SDS from the DNA. All these changes that were observed after the addition of these solutions were expected as they are what help us extract the DNA plasmid for an end product. (b) Agarose gel electrophoresis After placing the DNA plasmid in the wells electrophoresis was carried out. The results were then obtained and recorded. The size of the DNA fragment is determined from its electrophoretic mobility. The DNA fragments of know molecular weight markers are run on the gel and a graph of log MW against migration distance is drawn. There are three different forms of agarose DNA first theres the open circular plasmid DNA this is the first band that occurs on the picture. The circular plasmid is a  double-stranded  circular  DNA  molecule  that has been nicked in one of the strands to allow the release of any super-helical turns present in the  molecule. The open circular plasmid migrates more slowly than a linear or super-coiled  molecule  of the same size this is due to associated differences in  conformation, or shape, of the  molecules. this is why it is the first band that occurs on the picture result. Linear DNA has free ends, either because both strands have been cut, or because the DNA was linear  in vivo. The rate of migration for small linear fragments is directly proportional to the voltage applied at low voltages. At a specified, low voltage, the migration rate of small linear DNA fragments is a function of their length. Large linear fragments (over 20kb or so) migrate at a certain fixed rate regardless of length. This is because the molecules resperate, with the bulk of the molecule following the leading end through the gel matrix.  Restriction digests  are frequently used to analyse purified plasmids. These enzymes specifically break the DNA at certain short sequences. The resulting linear fragments form bands after  gel electrophoresis. It is possible to purify certain fragments by cutting the bands out of the gel and dissolving the gel to release the DNA fragments. This is neither fast nor slow in comparison to the other DNA plasmid. The super-coiled Plasmid DNA normally occurs naturally, there is super-coiling in DNA only if there is a replication of a DNA plasmid and this occurs for a small space of time and that is removed by cutting the DNA by specific enzymes, this is part of DNA replication mechinary. This type of DNA plasmid is the fastest as it is the last band shown out of the three this is Because of its tight conformation. The picture above shows the results obtained from the agarose gel electrophoresis. The lane numbers are marked over the wells. The lane before lane 1 that is titled M is the molecular weight marker. All three forms of plasmid DNA is present in this result, the open circular, the linear and the supercoiled. There is an extra band of RNA present however not clearly visible this is because the RNA fragments migrated ahead of dye front as diffuse a band, the ribonuclease gets rid of this band, a blue tracking dye cause the black smudge under the DNA plasmid and beneath that is the barley visable RNA. RNA is very unstable under these conditions, as a result RNA can be completely degraded befor the ribonuclease has been added. It can be seen that DNA is present more in one band then another, however the one with the less amount could have a bigger fragment. There seems to be logarithmic relationship between the size of the DNA fragment and the distance it travels on the gel. A standered curve can be made if we measure the length the bands in different lanes travelled if the fragment sizes are known. The more points plotted and the larger the separation there is on the gel, the results will be more accurate. Conclusion The experimental procedures carried out were a success, the DNA plasmid was obtained and the agarose gel electrophoresis resulted with in a clear picture as shown and outlined above, of the DNA being successfully separated. The uses of purified plasma in DNA research is for molecular cloning.

A Streetcar Named Desire, by Tennessee Williams Essay -- A Streetcar Na

A Streetcar Named Desire From the beginning, the three main characters of Streetcar are in a state of tension. Williams establishes that the apartment is small and confining, the weather is hot and oppressive, and the characters have good reason to come into conflict. The South, old and new, is an important theme of the play. Blanche and her sister come from a dying world. The life and pretensions of their world are becoming a thing of memory: to drive home the point, the family mansion is called "Belle Reve," or Beautiful Dream. The old life may have been something beautiful, but it is gone forever. Yet Blanche clings to pretensions of aristocracy. She is now as poor as Stanley and Stella, but she cannot help but look down on the humble Kowalski apartment. Stanley tells her that she'll probably see him as "the unrefined type." The differences between them, however, are more complex and volatile than a matter of refinement. Desire is central to the play. Blanche is unable to come to terms with the force of her own desire. She is clearly repelled and fascinated by Stanley at the same time. And though she stayed behind and took care of the family while Stella ran off to find a new life, Blanche is both angry and jealous of Stella's choice: she seems a bit fixated on the idea of Stella sleeping with her "Polack." Stella has chosen a life built around her powerful sexual relationship with Stanley. Blanche is both repulsed by and jealous of the choice. . The play is haunted by mortality. Desire and death and loneliness are played off against each other again and again. The setting is one of decay; the dying Old South and the dying DuBois family make for a macabre and unsettling background. Blanche's first monologue is a rather graphic description of tending to the terminally ill. There is also the specter of Blanche's husband, who died when they were both very young; indeed, Blanch still refers to him as a "boy." Another symbol is the meat: Stanley enters carrying a package of bloody meat, like a hunter coming home from a day of work. Stanley is a superb specimen of primitive, unthinking, brutal man. The meat-tossing episode is seen as humorous by Eunice and the Negro Woman, who infer a sexual innuendo from the incident. Apparently, it is obvious to the neighbors that the sexual bond between Sta... ...us line is full of terrible irony. It is true that Blanche has often depended on the kindness of strangers, but all of them have abused and abandoned her. In the end, even her own sister has betrayed her. Her fragility, her inability to fend for herself, and her self- deception have brought her to madness. The representative of the new man, Stanley, is more ape than knight. But Blanche's line is earnest in that it shows her terrible loneliness. For so long, she has known only strangers; young girl in a house full of the dying, and then a woman losing her looks seeking protection from callous men. Her tragedy will for the most part be forgotten. Stella is crying, but she has nonetheless decided to stay with Stanley. She also will have to busy herself with caring for the baby. The other men have callously chosen to go on with their poker game on this day, denying Blanche the dignity of being taken away in private. The Old South dies, and the New South does not mourn her passing. Everyone is going to move on: as the play ends, Steve is already dealing a new hand. Sources:Streetcar Named Desired by Tenesse Williams Northon Anthology www.Sparknotes.com www.classicnotes.com

Should Boxing Be Banned?

What do Boxing, Sky Diving, Horseback Riding, and Climbing Mountains have in common? All of these sports can be very dangerous to perform some more than others but dangerous all the same, yet thousands of people continue to attempt these sports and many more dangerous activities evry day. An obvious question somebody might ask is why doesn’t anyone stop these people from doing these sports if their so dangerous? The simple answer is, its nobody else’s business what other people do. You can not tell someone else what they can and can not do, that’s a violation any persons rights. For example, many people think boxing should be banned because it imposes danger to a body’s health, but I on the other hand do not think it should be banned at all, merely reformed in some rules and other components in boxing such as safety and fighting techniques. Many boxing regulations are designed to keep the boxers as safe as possible. Although the main objective is to basically harm your opponent, it is actually more complicated than that. There are many rules about gear, safety, injuries and fouls. For instance, boxing gloves and headgear are designed to cushion impact when you hit somebody or get hit. Grease is also used because it helps punches slide off wherever you have gotten hit. Matches are no longer a minimum of 15 rounds, they can be stopped at any time during the fight if a referee deems a boxer unable to fight any longer. Also, all coaches and referees must attend 2 yearly meetings to take exams and learn more about the safety guidelines of boxing. If they do not pass these exams then they can not participate in boxing or be involved in any way except for being a spectator to this sport. (Canadian Boxing Association 1) Many people also believe that amateur boxing is the same as professional boxing, but people involved in boxing would know that that’s not true. Amateur boxing is one of the worlds most regulated sports ever, therefore there are less severe injuries. Studies have shown that 30% of kids ages5 to 14 have been seriously injured at least once in their lifetime due to sports, but less than 1. 7% of those injuries were from amateur boxing. Also less than 4. 2% of children diagnosed with Mild Traumic Brain Injury receive these injuries because of either football, soccer or baseball but not boxing. Now professional boxing is slightly different from amateur in that once a match has begun it can not be ended unless a boxer to T. K. O or has forfeited. Moreover, professional boxing is seen as more of a business and concerns for bigger paychecks are very high. It is for these reason and many more that some people think or say that amateur boxing should be the only kind of legal boxing allowed in any country. Pena 1) As indicated earlier, there are other dangerous sports in this world and boxing isn’t nearly the most dangerous one, so why is only boxing being proposed for banning? The answer to this question no one is really sure about, but one thing we are sure of is out of 100,000 deaths or fatal injuries due to sports boxing is only responsible for about 1. 3% of that 100,000. The British Medical Association, howeve r, states that the harm inflicted by boxing in caused purposefully, well it generally is , and that the harm inflicted by other dangerous sports are only accidental, which may or may not be true, nobody knows for sure. But, in the long run, its really nobody else’s business what you do for a living or what you do in your spare time, if you love doing something nothing can stop you from doing it every day if you wanted. Not even the government or state can have a say in which sports you choose to participate in, the only thing they can do really is alter or change the rules and safety guidelines of the sport. In boxing there have already been major changes over the past decades. For example, ringside medical care has been more actively enforced ten-fold just over the past 3 years. Also, a licensed doctor and several paramedics are kept on sight at all boxing matches with a ready ambulance and any tools or instruments that are needed for a surgery, if one is required immediately. The more radical ideas about changing boxing are less admired by the public eye. For instance, there have been rumors about professional boxers being required to wear headgear, much like amateur boxers do at every match, and restricting blows to your opponents head or face area. These rules if actually enforced would indeed help protect the boxers way more, but in a way would reduce the appeal of the sport to many people. Wither way though, with or without headgear and the banning of blows to the head or not, injuries are bound to happen in any way shape or form. Many of these injuries like Parkinsons, Chronic Brain Damage or Mild Traumic Brain Injuries are not caused by just one match of boxing. These injuries are only accumulated over a boxers career or lifetime. So realistically it would be a long time before a boxer would get any serious or fatal injuries. Still there are people who believe that banning boxing would be a good thing to do. There are many groups, associations and even countries who campaign to end boxing for good. Some of the countries that actually banned boxing are Norway and Sweden. One of the more bold associations, the British Medical Association, have been campaigning against boxing since 1982, which is over 25 years. The BMA has been against boxing for so long because they think that the sport is very dangerous and makes violence look like an OK thing to do. The BMA proposes that the first step to getting rid of boxing for good is to first ban boxing At amateur level, which would be banning it for children ages 7 to 16, because they believe that boxing at this young an age is also very dangerous more than boxing at a professional age and that iut exploits children by placing them in rings and allowing them to fight one another. (Laurance 1) Likewise there are many doctors’ who agree that boxing should be banned due to the serious nature of the injuries. They believe that there is absolutely no safe level of boxing, that any hit or blow can contribute to complications of the brain and other main organs or even death. These doctors conclude that putting an end to this degrading spectacle as they call it would put an end to deaths, injuries, and brain damage caused by boxing. It could also end hundreds of young men and boys putting their lives at risk every day just for some sport. (Laurance 2) With all these reasons to ban boxing, some people would think that there are no positive outcomes or pros of this sport, yet there is. There are also many reasons why banning boxing would be a bad thing to do. One of the many motives for keeping boxing around is tat if boxing were made illegal it would only send the sport underground much like dogfights and chicken fights. I’m not saying that dogfights and chicken fights should be made legal because that’s just wrong, but I am saying that once boxing goes underground its going to be very hard to keep safety rules and regulations a part of the sport like they are now. There would also be no licensed or legal doctors on sight to help fighter as soon as possible when they get injured. It was also proven that 70 years ago, when boxing was taught in school, that society was less violent and people were less aggressive against each other. Boxing teaches people self control over their aggression and tempers in bad situations, which is very helpful for everyone. There are many benefits to boxing, as well,

Manifest Destiny Research Paper - 1382 Words

Samantha Mooney English 11B Travis DeVore April 13th, 2014 Manifest Destiny Research Paper: The 1840’S were years of unprecedented growth for United States; in a mere four years, the national domain more than doubled with an additional 1.2 million miles being added to the country. (PBS) This was due to a movement called Manifest Destiny that suggested that the United States was â€Å"destined† to stretch from coast, sea to shining sea, uninterrupted by anything or anyone. (History.com) However, complex and underlying motives guided Manifest Destiny advocates. Some believed that the nation had a divine order, serving as a bastion of Christianity and Democracy in a land of uncivilized Native Americans. Ideas of the Anglo-Saxon race being a superior race permeated as well. Others saw economic or national security concerns dictating expansion as a necessity. As if the varying motivations didn’t complicate the issue enough, Manifest Destiny was more of a movement than an event. There is truly no start or end date, rather it i s a coalescing of various factors that will be discussed throughout this paper. Therefore this paper will seek to explain the ideas, actions, and reactions to Manifest Destiny by differing individuals. Since Manifest Destiny has no specific starting point, it is best to understand the origin and meaning of the term. Newspaper writer John Sullivan coined it when he wrote â€Å"[it is]†¦the right of our manifest destiny to†¦spread and to possess the whole of theShow MoreRelatedThe Main Events And Struggles Of The War With Mexico1385 Words   |  6 Pagesyour research on your topic. It is not an introductory paragraph. It should be three to four sentences in length. The Main Events and struggles of The War with Mexico in 1846-1848 Your introductory paragraph starts here. It is an explanation of what or who your subject is. We will also talk about what events led to the war. 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