Molecular cloning

Molecular cloning Abstract Molecular cloning is a method to produce quantities of a specific DNA segment. It contains an abundance of techniques including DNA transfer, DNA recombination, DNA sequencing and so on. Though this practical, restriction maps were draw for plasmid pMA and pMB by doing single and double digest, a pMB fragment digested with PstI was inserted to plasmid pUC19 and then transferred to host cells to have proliferation and expression, and the sequence of PstI digested pMB fragment was analysed. 1. Introduction Recombinant DNA molecules are molecules containing DNA sequences derived from more than one source. In molecular cloning, by using recombinant DNA, a specific combination of genes can be put into a carrier, and then can be proliferated and expressed in a recipient cell. In medicine, by making use of molecular cloning, scientists have successfully constructed engineering strains of insulin, growth hormone of human, cattle and chicken, human interferon, erythropoietin, antigen of hepatitis B virus and antigen of foot-and-mouth disease virus, and conducted a large-scale production by fermentation industry. In gene therapy, there is a possibility of reversing cancer cells to normal cells through genetic engineering, for example, mouse tumor cells caused by SV40 virus can reverse to normal cells at high temperature. Many chemical reagents such as acrylic acid, ethylene glycol, methanol, ethylene oxide and salicylic acid can possibly be produced by making use of molecular cloning. In environmental protection, people transfer genes of one microorganism into another through genetic manipulation to create new strains that are more capable of degrading harmful substances, in order to break down toxic substances in industrial waste.[1,2] Blue-White selection is a method for screening recombinant DNA. Vectors containing a ÃŽ ²-galactosidase gene (lacZ) can have a complementation (ÃŽ ±-complementation) with E.coli strain to form a functional ÃŽ ²-galactosidase enzyme. Neither vectors, nor host cells have the enzyme activity. The lacZ gene has an internal multiple cloning sites (MCS) which can be cut by different restriction enzymes. Therefore, when a gene fragment is inserted in the vector, the lacZ gene will be disrupted and cannot form active ÃŽ ²-galactosidase enzyme. X-gal can be metabolized by ÃŽ ²-galactosidase to gain a blue product. Therefore, in the presence of X-gal, DNA with no insert gene can display a blue colour, while recombinant DNA, which have no enzyme function, display a white colour.[3] The aim of the practical is to draw restriction maps of simple plasmids for recombinant DNA, do basic molecular cloning and sequence a DNA fragment. 2. Results Table 1: Antibiotic resistances of 5 E. coli strains LB/Ampicillin LB/Tetracycline LB/Kanamycin DH5a No growth No growth No growth pUC19 Grown No growth No growth pMA Grown Grown No growth pMB No growth Grown Grown XL1-Blue No growth Grown No growth DH5a: E. coli strain DH5a; pUC19: E. coli strain DH5a containing plasmid pUC19; pMA: E. coli strain DH5a containing plasmid pMA; pMB: E. coli strain DH5a containing plasmid pMB; XL1-Blue: E. coli strain XL1-Blue. NO. DNA Enzyme 1 pMA Bam HI 2 pMA XhoI 3 pMA PstI 4 pMA EcoRI 5 pMB Bam HI 6 pMB XhoI 7 pMB PstI 8 pMB EcoRI 9 Lambda marker 10 X174 marker NO. DNA Enzymes 1 pMA EcoRI, Bam HI 2 pMA EcoRI, PstI 3 pMA EcoRI, XhoI 4 pMA Bam HI, PstI 5 pMA Bam HI, XhoI 6 pMA PstI, XhoI 7 pMB EcoRI, Bam HI 8 pMB EcoRI, PstI 9 pMB EcoRI, XhoI 10 pMB Bam HI, PstI 11 pMB Bam HI, XhoI 12 pMB PstI, XhoI 13 Lambda marker 14 X174 marker 1 Lambda marker 2 Blue colony digested with PstI 3-7 White colonies digested with PstI 8 X174 marker gagtantagttcgccngttaatagtttgcgcaacgttgttgccattgctgcaggggggggggggaaagccacgttgtgtctcaaaatctctgatgttacattgcacaagataaaaatatatcatcatgaacaataaaactgtctgcttacataaacagtaatacaaggggtgttatgagccatattcaacgggaaacgtcttgctcgaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgcctcttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccccgggaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataagcttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttgatgaggttatttttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttttaatgagaattggttaattggttgtaacactggcagagcattacgctga cttgacgggacggcggctttgttgaataaatcgaacttttgctgagttgaaggatcagatcacgcatcttcccgacaacgcagaccgttccgtggcaaagcaaaagttcaaaatcaccaactggtccacctacaacaaagctctcatcaaccgtggctccctcactttctggctggatgatggggcgattcaggcctcaacgactgagtatggaccttcttcacgaggcagacctcagcgccccccccccccctgcaggca Enzyme No. of cuts Position of sites (bp) Recognition sequence PstI 2 52, 1243 ctgca/g XhoI 1 204 c/tcgag E X Stop F A X Stop _ F A Q R C C H C C R G G G E S H V V S Q N L Stop C Y I A Q D K N I S S Stop T I K L S A Y I N S N T R G V M S H I Q R E T S C S R P R L N S N M D A D L Y G Y K W A R D N V G Q S G A T I Y R L Y G K P D A P E L F L K H G K G S V A N D V T D E M V R L N W L T E F M P L P T I K H F I R T P D D A W L L T T A I P G K T A F Q V L E E Y P D S G E N I V D A L A V F L R R L H S I P V C N C P F N S D R V F R L A Q A Q S R M N N G L V D A S D F D D E R N G W P V E Q V W K E M H K L L P F S P D S V V T H G D F S L D E V I F D E G K L I G C I D V G R V G I A D R Y Q D L A F S K I W Y Stop _ S Stop Y E Stop I A V S F D A R Stop V F L M R I G Stop L V V T L A E H Y A D L T G R R L C Stop I N R T F A E L K D Q I T H L P D N A D R S V A K Q K F K I T N W S T Y N K A L I N R G S L T F W L D D G A I Q A S T T E Y G P S S R G R P Q R P P P P C R Aminoglycoside 3-phosphotransferase, putative 3. Discussion 3.1 Antibiotics resistances Seen from table 1, DH5a has no resistance to any of the three bacteria, pUC19 is resistant to ampicillin, pMA is resistant to ampicillin and tetracycline, pMB is resistant to tetracycline and Kanamycin, and XL1-Blue is resistant to tetracycline. Plasmid pUC19, pMA and pMB, which were used in the cloning procedure, had different antibiotic resistances, while the bacterial host, DH5a, have no antibiotic resistance. Therefore, cells containing recombinant DNA could be selected by growing host cells in presence of antibiotic. Even when different plasmids are contained in the host cells, this method can be used. For example, tetracycline can be used to select cells containing only pMA from a mixture of cells containing pMA and pUC19. 3.2 Restriction maps and relationship of pMA and pMB From the single digest (1), pMA could be cut by Bam HI, PstI and EcoRI, and each enzyme could cut pMA once. However, pMA could not be cut by XhoI. pMB could be cut by Bam HI, XhoI and EcoRI once, and cut by PstI twice. Therefore, pMA has three restriction enzyme sites, while pMB has five. From the double digest (2), the results were consistent with single digest, and the length of each fragment could be obtained. Restriction maps (3) were drawn based on the single and double digests. From the restriction maps, the fragments in pMA and pMB, cutting by Bam HI and EcoRI, have the same base pairs (430bp). The fragment cutting by EcoRI and PstI in pMA has the same base pair (720bp) with one of the fragments cutting by EcoRI and PstI in pMB. The fragment cutting by Bam HI and PstI in pMA has the same base pair (1150bp) with one of the fragments cutting by EcoRI and PstI in pMB. The longer fragment in pMB cutting by PstI was round about 3780bp, which was very close to the length of pMA (3800bp). As all the lengths of fragments were roughly obtained and were not accurate. Therefore, we can assume that pMA is a part of pMB. pMB can be cut by PstI. If the longer fragment is re-circled, it will have the same base pairs and restriction enzyme sites (PstI, EcoRI and Bam HI) with pMA. The XhoI restriction site on pMB is between the two restriction sites of PstI, therefore, the longer fragment cannot be cut by XhoI, which is consistent with pMA. Seen from the antibody resistances, pMA is resistant to ampicillin and tetracycline, pMB is resistant to tetracycline and Kanamycin. This might because the tetracycline resistant gene is in pMA, which is a part of pMB. And kanamycin resistant gene is in the PstI fragment of pMB, which pMA does not have. For the ampicillin resistant gene, it might be located around the PstI restriction site in pMA, which will be insertion inactive when insert the PstI fragment to pMA to make it become pMB, therefore, pMB does not have ampicillin resistance. This hypothesis can be proved by sequencing pMA and pMB fragment cutting by PstI, which was not included in this experiment. 3.3 Sub cloning recombinant clones In 4, the blue colony had only one band, which meant that there was only one PstI restriction site in the plasmid. This was consistent with pUC19 that did not have an insert fragment. Four of the white colonies had two bands each, including one band located around 1200bp. These were the recombinant DNA, with the pMB fragment digested with PstI. One white colony (No. 7) did not have a band located around 1200bp, but a fragment shorter than that. This was also a recombinant DNA, with other fragment rather than PstI fragment. This might be caused by some impurities through the procedure. 3.4 Sequence analysis The sequence of the PstI fragment in pMB was obtained by overlapping two fragments (forward and reverse). Seen from 5, there are two PstI restriction sites (ctgca/g) and one XhoI restriction site (c/tcgag), and the XhoI restriction site is between the two PstI restriction sites. Therefore, if the fragment is digested with PstI and XhoI, two fragments (152bp, 1039bp). This is roughly consistent with the restriction map of pMB which was not accurate. The amino acid sequence shown in 6 is one of the six possible sequences (53 Frame 1), methionine, which is a start of protein sequence, and stop codons are over striking. One potentially matching sequence of protein encoded in the PstI fragment of pMB shown in 7, aminoglycoside 3-phosphotransferase, begins with the first methionine in the fragment and have a length of 253 amino acids. 4. Conclusion This practical provide us a better understanding of how to make a recombinant DNA and molecular cloning technique. These experiences can act as fundament of further researches such as researches in cancer cells. References: [1] Williams Wu, Michael J. Welsh, [et al.] (2003) Gene Biotechnology (2nd edition). [2] Gerald Karp. (2002) Cell and Molecular Biology (3rd edition). [3] Benjamin Lewin. (2004) Gene (International edition).

How does Russell make the audience sympathise with Shirley ? Essay

In this essay I will be exploring how Willy Russell, an author of the play â€Å"Shirley Valentine†, makes us sympathise with his main character. Willy Russell was a hairdresser, who lived in Liverpool. He felt unfulfilled in his life and wanted to become a writer. In his work, he met a lot of women, who shared their stories with him. That made him feel he understood many of them. But he was bored with his profession. Russell’s life experience is similar to Shirley’s. Just like himself, Shirley is lonely and unfulfilled in her life. She dreamed of going to Greece and sitting alone on the beach. Russell equally dreamed of something unattainable. They both are bored and disappointed with their everyday routine. Shirley, a middle aged woman, is disappointed because her husband treats her as a housewife and she does not have a life outside of the house. No one pays any attention to her and she feels underappreciated. Everyday life looks exactly the same. Russell uses a number of techniques to make us sympathise with Shirley. These include providing a social context for the play, flashbacks, language devices such as humour, dramatic monologues and voice-overs. The play is about a middle aged woman called Shirley Valentine. She is married to a man called Joe. When they newly was married they loved each other so very much, but after a few years her life became a routine of washing plates, dishes, cleaning and making food for her husband. In other words she became a housewife. Because she had nobody to talk, she is a Kitchen sink drama, it is about things around the house. Russel also used characters, dramatic devices and creative language to sympathise the audience with Shirley Valentine. In the opening scene we see first the film title track starts under a blue and white drawing of Shirley Valentine ironing. I think Willy chose the colour blue, because the colour blue suggest sadness and depression. Shirley’s life in the play is represent as sadness and depression. After that we see fifteen drawings of Shirley doing domestic works are never ending. None of the drawings show her having fun. Russel did this to show that her life is around her house. Then we see a drawing of a street of small semi-detached houses with small front gardens. This show where she lives. She turns in one of the houses and opens the front door. She closed the kitchen door and leans against it â€Å"Sighing† . This show she is fed up with her life maybe she is frustrated. â€Å"She talks to the wall† that shows she is lonely. She have nobody to talk with and it suggest she might going to be crazy. Russel uses the characters of Joe to make us sympathise with Shirley Valentine. He is Shirley Valentine’s husband. We sympathise with Shirley as Joe doesn’t show any love affections towards her. We can tell Shirley was happily married at the start, because she had fun with Joe. They was just newly married. â€Å"The are happily painting the kitchen†. This shows us that she was happy with her life. Joe was Romantic used to make her laugh. He told her that he love’s her, â€Å"I love you Shirley Valentine†. But now he is extremely changed. He is aggressive. This shows where he said he doesn’t care what Shirley is doing. That’s why Shirley take a decision to go too Greece. There she meet Costas. Costas is the opposite of Joe. He is Romantic and he listening to her, he made her feel warm and tells her how beautifull she is. â€Å"They are lovely because they are part of you and you are lovely. Joe never told her how beautifull she is Costas make her feel pretty and young.

The Dos and Donts of Biotechnology Essay Topics

The Do's and Don'ts of Biotechnology Essay Topics What's Really Going on with Biotechnology Essay Topics Biotechnology can enhance a animals impact on the surroundings. Although it seems to be appealing, it is also highly controversial. Most importantly, it is needed to feed the growing population of the world, especially in Third World Countries. Agricultural biotechnology was used to safeguard crops from devastating diseases. Industrial is also a sub-discipline that is connected to the biotechnology market. As stated by the research, most biotechnology firms depend on venture capital funding to a larger extent. Biotecnika is the Only biotechnology platform which gives biotechnology work in real biotech businesses. Biotechnology Essay Topics Can Be Fun for Everyone On the other hand, the isolation of this sort of single molecule treatment caused the growth of quinine-resistant malaria. There is really a moral issue around telling people they may develop certain illnesses later on. This produces enormous difficulties. Don't neglect to explain why the issue is significant to you! Section Editors commission reviews from authorities on each and every topic that they've selected. It's possible to write on the next topic to acquire big score in your dissertation. Thus, I would attempt to answer this based on the present hot topics and my private region of interest. It made them learn the fundamental breeding practices. An intriguing one may be the effect capitalism and other systems has had on the subject of biotechnology generally. Many other kinds of crops are presently in the research and development stages. You may upload this file from this site for your submission. If you're confused with a number of interesting topics to research on the web, it's far better to choose what interests you the most. Stop by the AC21 page to find out more. For extra information please see the appropriate agency sites. Life After Biotechnology Essay Topics Application essays about challenges reveal how you respond to difficulty to folks who are rather interested in how you'll handle the subsequent four years all on your own. But prior to obtaining a profession on this subject an individual has to experience several research studies together with thorough comprehension of the subject. You're attempting to show colleges your very best self, therefore it might appear counterintuitive to willingly acknowledge a time you struggled. So whether you're looking for discursive essay topics on business studies or science everything can be found from the site of StudentsAssignmentHelp.com. There is quite a modest difference between the deductive and argumentative essay and that is the reason why it is quite easy to prefer these topics for argumentative essay and persuasive essay too. All persuasive essays are like argumentative essays. There are many intriguing topics that could be become a persuasive essay if you take the opportunity to think about doing it. They have to ask questions too. With FreeEssayHelp you'll find hundreds of Biotechnology essay topics in a matter of many seconds. Alongside the topics, you'd discover loads of papers free of charge. Ideas, Formulas and Shortcuts for Biotechnology Essay Topics But as scientists become adept at deciphering somebody's genetic composition, it's increasingly possible that compromising information about an individual's future health will turn into available. The area of medicine has come a very long way from using heroin for a cough remedy or magnet therapy to enhance the flow of blood. Their patient was prepared to go home. They need to take care of the recent findings and debatable questions. We're discussing a costly insurance policy and, like every insurance policy, you hope you don't ever have to utilize it, states Krause. Colleges are searching for a feeling of maturity and introspectionpinpoint the transformation and demonstrate your private growth.

Essay on Robert Ressler Coined the Term Serial Killer

â€Å"We serial killers are your sons, we are your husbands, we are everywhere. And there will be more of your children dead tomorrow.† (Ted Bundy). Serial killers are not always those people that look like monsters or behave in strangeous ways. Sometimes they are the successful people, the ones that have a family and a job. The term â€Å"Serial Killer† was first coined by Robert Ressler, former director of the FBI’s Violent Criminal Apprehension Program. Serial killers are often defined as people that kill two or more people over a period of more than 30 days with â€Å"cooling off† periods between each kill. Many historical criminologists suggest that serial killing has been a component of society since the beginning; suggesting that old stories†¦show more content†¦The triad includes fire-setting, animal cruelty, and enuresis (bed-wetting). A highly debate issue around serial killers is the reason behind their actions. Although many theories exist about the reason why serial killers act the way they do, the most common theory is that they are searching for some type of psychological satisfaction. A very common theory behind the reason of the actions of serial killers is the social control theory. This theory states that serial killers behave the way they do due to their inability to cope and adapt to society. Another well-known theory is the neutralization theory. This theory states that murderers sometimes move from an invisible â€Å"baseline† and commit illegal actions while sometimes moving back and acting in a conventional way. The last theory is the labeling theory. This theory, created by Erving Goffman, states that people act the way they do due to the labels that society has created for them. Therefore, if a kid is labeled as a troublemaker at a very young age, it is more likely that he will continue with this behavior through adulthood. From Ted Bundy to BTK to the â€Å"Zodiac Killer†, serial k illers have been around the United States for a long time, but none is better than Richard Chase, or better known as the â€Å"Vampire of Sacramento†, to investigate the reasons behind serial killers. Born on May 23, 1950, Chase was raised in a very strict house and was constantly abused by his parents. As aShow MoreRelatedSerial Murder And Mass Murder936 Words   |  4 Pages Before we can take on the definitions of serial murder and mass murder, we must first understand what exactly constitutes murder. According to the United States Code-section 1111, murder is defined as the unlawful killing of a human being with malice aforethought (4). With that said, according to the Federal Bureau of Investigation, the term serial murder implies that there are at least three different murder events at three different locations, with a â€Å"cooling off† period between each event (RamslandRead MoreThe Crime Of Serial Murder And Mass Murder1289 Words   |  6 PagesRotten Apples The act of Serial murder and mass murder have similar characteristics, however they are not the same. Before they can be defined, it is necessary to first understand what exactly constitutes murder. According to the United States Code-section 1111, murder is defined as the unlawful killing of a human being with malice aforethought (FindLaw, 2014). With that said, according to the Federal Bureau of Investigation, â€Å"the term serial murder implies that there are at least three different