Mandatory Ethnography

Initially, the DNA of interest needs to be isolated to provide a DNA segment of suitable size. Subsequently, a ligation procedure is used where the amplified fragment is inserted into a vector . The vector is linearised using restriction enzymes, and incubated with the fragment of interest under appropriate conditions with an enzyme called DNA ligase. Following ligation the vector with the insert of interest is transfected into cells. A number of alternative techniques are available, such as chemical sensitivation of cells, electroporation, optical injection and biolistics. Finally, the transfected cells are cultured. As the aforementioned procedures are of particularly low efficiency, there is a need to

The vital components and techniques of gene cloning are as follows, the DNA sequence that contains the desired gene (EZH2) is amplified by Polymerase chain reaction. PCR was established by Kary Mullis in 1985, popularly known to amplify target sequences of DNA (EZH2) to a billion fold in several hours using thermophilic polymerases (Taq) ,primers and other cofactors (Sambrook and Russell, 2001). Three crucial steps are involved which are Denaturation (at 95°), Annealing of the forward and reverse primers (55-65°) and lastly primer extension (at 72°). After amplification the desired sequence is integrated into the circular vector (pbluescript) forming the recombinant molecule. For the compatibility of the insert and vector, both were digested with (EcoR1) so the same cohesive ends are generated in both, making it easier to ligate. EcoR1 is a restriction enzyme that belongs to the type II endonuclease class which cuts within dsDNA at its recognition site “GAATTC” (Clark 2010; Sambrook and Russell, 2001).

In this investigation pUC19 plasmids were used as the vector due to its small size of 2686bp, high uptake efficiency by the host and fast replication time. Important features of this plasmid include the origin of replication and multiple cloning sites (MCS). The origin of replication allows the plasmid to replicate inside the host bacterium. The MCS is located within the lacZ gene and contains unique sites for the Xbal & EcoRI restirction enzymes to cut and produce sticky ends for the CIH-1 gene to bind to. Furthermore, the pUC19 plasmid also contains an ampiccilin resistance gene so only transforemed E.coli are able to remain viable when spread on the agar plates that also has the addition of ampiccilin. The lacZ gene encodes the β-galactosidase enzyme which aids in indentifying the recombinant E.coli from the non recombinant cells (Coventry University 2016).

Throughout analysis of the data, we complete DNA agarose gel electrophoresis in order to determine the size of our plasmid DNA. The electrophoresis

The purpose of the experiment was to isolate plasmid DNA, followed by restriction digestion using restriction endonucleases and then visualizing the digested fragments after subjecting to gel electrophoresis. Plasmid DNA (pSP72 DNA) was isolated from Escherichia coli KAM32 (E.coli) cultures using the QIA prep miniprep kit and then subjected to restriction digestion by EcoRI and HindIII. The restriction digested DNA was then loaded into the wells of 0.7% agarose gel and subjected to electrophoresis. It can be concluded from our results that our plasmid DNA isolation was successful and the restriction digestion results were partially in agreement with our hypothesis.

Queens is the most diverse borough in New York City, and one of the most diverse communities anywhere. To what Cultural or ethnic communities do you belong? Is there an art work (it can be a song, a piece of fine art, a dance or a performing art piece) that has a special significance in your culture? What is it and what does it signify to you?

Analysis of DNA from practicals 1 and 2 using the technique of agarose gel electrophoresis and analysis of transfomed E. coli from practical 2 (part B)

The ethnography I chose to read and write about is “How Musical is Man?” by John Blacking. John Blacking is a famous British anthropologist but he later got trained to be an ethnomusicologist and was a major influential musical figure in history. He often thought that music was “humanly organized sound” and that “no musical style has its own terms but more of the terms of its society and culture. Throughout the book, Blacking made many great points and teaches us in a nonbiased way. In this book, John Blacking discusses the nature of musicality, the role of music in society, learning, and the musical discourse. “How Musical is Man?” is an ethnography on the music of the Venda people of South Africa and how each society views and experiences it.

Pure plasmid pET28a, amplified 2-alcohol dehydrogenase gene (a PCR product), 10x bovine serum albumin, 10x neutralization buffer, EcoRI, nuclease free water, pET28a plasmid digested with EcoRI, calf intestinal alkaline phosphatase, agarose gel (1% agarose + 0.3μL ethidium bromide), 1x TAE buffer (40mM Tris base + 0.1% acetic acid+ 1mM EDTA), pET28a plasmid digested alkaline phosphatase and EcoRI, DNA ladder, 6x loading buffer (30%glycerol + 0.25% bromophenol blue), parafilm, n-butanol TE buffer, 95% and 70% ethanol, TE buffer (10mM Tris-Cl, pH 7.5) and 1mM EDTA were purchased from Sigma-Aldrich. Micropipettes, eppendorf tubes, eppendorf master cycler gradient, Labline orbit shaker, So-Low ultra low freezer and AvantiTM J-301 centrifuge.

After the incubation, 1.5 mL of each of the three cultures were added to eppendorf tubes and centrifuged at 13,200 rpm for 1 minute. An alkaline lysis procedure like that of Birnboim and Doly was then performed to extract the plasmid DNA with 200 μl of alkaline SDS detergent solution (Birnboim & Doly, 1979). After

Amplified luxAB genes of isolated chromosomal DNA from Vibrio fischeri bacteria were ligated into digested pGEM [1]. The cut pGEM contained a “stuffer” – a small fragment of DNA positioned between the EcoR1 and Xba1 restriction sites and located within the polylinker region of the plasmid [1]. The stuffer fragment could potentially re-ligate back into the plasmid instead of the desired insert, namely the digested PCR product with luxAB genes [1]. Ligation reactions varied in two ways: via insert to plasmid ratios (either 1:1 or 2:1) and via the inclusion or exclusion of the stuffer fragment within pGEM [1]. The purpose of this experiment is to assess the effects of varying ligation parameters on the success of cloning luxAB genes

Restriction fragment length polymorphism utilizes restriction endonucleases and gel electrophoresis to confirm the similarity of several DNA samples. Fragments formed by restriction digest of the DNA molecules of several samples are run on an agarose gel; samples containing the same DNA will exhibit similar band patterns. For the investigation of an aggressive infectious disease caused by a new strain of bacteria that has acquired multiple resistance plasmids from other bacterial strains, restriction digest is performed on bacterial DNA samples isolated from patients stricken with the disease and on five plasmid samples using PstI and EcoRI. Agarose gel electrophoresis was executed for the six samples and the bands were compared and analyzed. The purpose of this experiment is to identify the bacterial strain responsible for causing the new disease using techniques described above. The crime scene sample contains fragments of the following molecular masses: 7992.82 bp, 4731.51 bp, 3767.04 bp, and 2676.09 bp, and the Suspect 3 sample contains fragments at the following molecular weights: 11251.2 bp, 7992.82 bp, 4624.88 bp, 3599.15 bp, and 2615.77 bp. Because four fragments of both samples are of similar molecular weights, Suspect 3 was identified as the bacterial strain responsible for causing the disease.

Hamilton and Armstrong conducted a long term ethnography on a group of college women throughout their college years, assessing and analyzing their sexual experiences. The main objective of this study is how gender and class shape sexuality for young college students. The authors mention that the women in the studied found hookups problematic as men were able to hookup without negative criticism while women would be called ‘sluts’. Therefore, the fear of the ‘slut stigma’ constrained a majority of these women’s sexual behaviors. The author mentions that many of the women wanted to have intercourse but waited until they had boyfriends in order to prevent getting called a ‘slut’. Furthermore, the sexual double standard legitimized negative treatment

800μL of sterile water and 200μL of the DNA isolate were pipetted into a 1mL UV-transparent curvette and mixed well. 1mL of sterile water was added to a second 1mL curvette. On the spectrophotometer, a baseline correction was done and the absorbance spectrum of the DNA isolate was recorded. A PCR master mix was made. 5μL of DNA isolate was pipetted into two new labelled PCR tubes. 5μL of Milli-Q water were pipetted into a third labelled PCR tube. 20μL of the created master mix was pipetted into one PCR tube and 20μL of the given master mix was pipetted into the other PCR tube. PCR tubes were placed in a thermocycler, and then frozen. Tube identification was noted. Electrophoresis Gel apparatus was set up using 1% molten agarose. 3μL of each of the three PCR tubes were pipetted into 3 labelled microcentrifuge tubes. 6μ of sterile water and 1μL of 10x loading dye was added to each. Gel was loaded with samples and a DNA ladder and gel was run. An image was captured and interpreted. Sample with the largest quantity of DNA, was prepared for sequencing. 10μL of

Plasmids were extracted using the three-step alkaline lysis procedure as outlined by Kado and Liu, 1999. The extracted plasmids were then digested using 1 U of each XhoI and XbaI, respectively according to manufacturer’s (New England Biolabs) recommendation and ran on a 1% Tris Acetate EDTA (TAE) agarose gel. 1 μl of BioLine Hyperladder I was used as the DNA marker.