Figure 1 - Ponceau Stain blot
Key :
1) Protein Molecular Weight Marker
2) Recombinant pET41(+) EGFP plasmid (positive control)
3) Non-recombinant pet41(+) EGFP plasmid (negative control)
4) Green colored clone properly expresses EGFP (green)
5) White clone that does not express EGFP (red)
6) Unknown sample (blue)
7) Purified GST-EGFP control
A Ponceau stain can bind and identify all proteins. Lanes 2, 3, and 4 (our recombinant, nonrecombinant and green colony, respectively) have a slightly smeared pattern of multiple bands that goes from 245 kDa to 80 kDa. Lanes 2 and 4 have faint banding patterns that descend from 80 kDa downwards. Lane 3 ends a bit early, around the 135 kDa mark. Lanes 5-7 (our white colony, unknown colony and purified
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protein control) have much less bands. 5 Has only a strong band at the top, between 190-245 kDa with some faint streaking ending before 80 kDa. The unknown sample has more proteins, with a huge smudging band between 135 and 100 kDa. Another band occurs around 90 kDa. The purified GST-EGFP control has only bands at the very top of the gel. Western Blot Figure 2 - Western Blot of E. coli colonies for EGFP detection Key: 1) Protein Molecular Weight Marker 2) Recombinant pET41(+) EGFP plasmid (positive control) 3) Non-recombinant pet41(+) EGFP plasmid (negative control) 4) Green colored clone properly expresses EGFP (green) 5) White clone that does not express EGFP (red) 6) Unknown sample (blue) 7) Purified GST-EGFP control The Western Blot looks only for a specific protein and would otherwise create no bands if the specific protein was not present. The protein ladder is in lane 1. Lanes 2 and 4 (the recombinant positive control plasmid and the noncontrol recombinant plasmid) display an intense amount of banding. Thick bands cascade from 200 kDa to 100 kDa, with an intensely thick band displaying itself right above the 100 kDa mark. The control recombinant bands are much cleaner at the top, but start to streak below the thickest band. The experimental recombinant bands are much thicker at the top, but thin out and streak less as they hit the bottom. The control and experimental nonrecombinant display no bands. The unknown sample (lane 6) has four bands at the 200, 160, 120 and 100 kDA marks with no streaking continuing below. The purified protein control (lane 7) has a thick band at the very top (245 kDa) with faint bands continuing all the way down. Two other notable bands are present, a faint one at 100 kDa (where the thick bands are in the recombinant and unknown samples) with a secondary fainter band at the 190 kDa mark. Affinity Chromatography Results Figure 3 - Affinity Chromatography results of supernatants, flow through, washes and elutions. Key: FT = Flow through SN = Supernatant W1-W10 = Washes, with the number of the wash next to the W. E1-E10 = Elutions, with the number of the elution next to the E. Each sample collected shows minimal glowing or reaction to the UV light. Green fluorescence should be more prominent, especially around the 3rd and 4th wash/elutions. The flow through has no glow, and the supernatant has minimal glow. Washes 1-4 seem to possess more EGFP, but washes 6 and 7 seem brighter. The elutions are the brightest out of everything here, but even then they are remarkably faint. Elution 3 seems to be the brightest thing here, with elutions 6 and 7 being similarly bright but containing much less EGFP. There appears to be minimal content in washes and elutions 8-10. Bradford Assay Results Table 1 - Bradford Assay Results The Bradford assay is meant to quantify how much protein is present in a sample. The Y-axis details the sample being used. The x-axis details the parameters of each sample. After the correlation was obtained from the standards (see Figure 3), the soluble protein concentration was calculated and then divided by the dilution factor to find the original concentration. This was multiplied by the total volume in ml to give a mass for the amount of soluble proteins. The mass of elutions was divided by the sum of the fractions to give a final percentage of 16.777% EGFP contained out of all proteins in the crude lysate. The absorbance rates are inconsistent but small as they go from Wash 4 to Wash 10. 60% of the washes were below the fourth, and lowest, standard. The supernatant absorbance rate should be lower, as it is not very reactive to the UV light. Unlike the washes, the elutions follow a bell curve pattern before lapsing into inconsistency once the numbers become small enough (around elution 7). Samples that were diluted with water returned with significantly stronger readings than those that did not. The strongest readings of the washes yielded the most result, but the elutions were more consistent with the quantity of EGFP they contain. Standard Curve for Bradford Assay Figure 3 - Standard Curve for Bradford Assay The Bradford Assay standard curve was calculated to have a y = 0.00888x equation.
The Standards, their concentrations and their absorbance are listed in the table below the graph. The x-axis is the concentration of the Bovine Serum Albumin, while the y-axis is the absorbance reading that was received when the spectrometer was set to 595nm.
Discussion
The data confirms that EGFP is present, but in abnormally small and low amounts. The Ponceau Stain confirmed that there are proteins in each sample, and the Western Blot confirmed the presence of EGFP in the samples that should have EGFP and confirmed the absence of EGFP in the samples that should not have EGFP. The unknown sample was revealed to have EGFP, therefore confirming its identity as containing a recombinant plasmid. However, the UV illumination post-affinity chromatography and the low readings on the absorbance meter indicates that EGFP is just barely present. With EGFP composing of ~17% of the crude lysate, it is possible that proteins are in very small quantities and thus could not be expressed definitively. The source of this error was more likely in the beginning of the lab, but the error cannot be traced with certainty as the original gels for ligations, transformations and digests were penetrated and could not yield at a discernable
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result. However, these results confirm the suspicions first aroused in the PCR test; that there is little EGFP in these samples.
The UV illumination after purification is faint and completely muted and the Bradford Assay absorbance readings were inconsistent in terms of pattern but still exceptionally small, which might contribute to the inability to get a precise reading. If the experiment was relaunched, samples with this small amount of EGFP protein should at first be checked without dilutions to see a rough estimation of how much raw protein is present. Had there’d been enough EGFP protein to create a successful result, elutions and washes 3 and 4 would glow considerably, along with the supernatant and flow through. All the elutions would also be brighter and more green. The absorbance readings and, as a result, the rest of the numbers, would also be higher and not fluctuate, especially as the frequencies were read for the last of the washes and the
elutions. Citations: Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. 1994. Green Fluorescent Protein as a Marker for Gene Expression. Science. Vol. 263. Pages 802-805. Obtained from science.sciencemag.org (http://science.sciencemag.org/content/263/5148/802?ijkey=440bdd5554399b2e491c5edb064fa9ec3fcc0933&keytype2=tf_ipsecsha) Retrieved 5/25/16 Lippincotz-Schwartz J, Patterson G. 2003. Development and Use of Fluorescent Protein Markers in Living Cells. Science. Vol. 300. Issue 5616. Pages 87-91. Obtained from science.sciencemag.org (http://science.sciencemag.org/content/300/5616/87.article-info) Retrieved 5/25/16.
The transducer in the assay was the Shimadzu UVmini-1240 UV-Vis Spectrophotometer. It is used to measure the absorbance of ferricyanide in solution. Ferricyanide is a yellow species that be measured and compared to the glucose concentration of the sample. Electrochemical glucometers look like the most common type of transducer for commercial use. It utilizes electrodes and flowing current measured by a voltmeter.2
Absorbance was defined as: log I_o/I where I_o is incident light and I is the transmitted light. Fluorescence emission spectrum is different from fluorescence excitation spectrum because it records different wavelengths of chemical s...
Ligation of EGFP into pET41a(+) vector transformed into E. coli cells followed by PCR amplification of extracted DNA plasmid for success evaluation along with gel electrophoresis at each step.
Digestion of the haemolytic and non-haemolytic cells allowed for easier identification of fragments during electrophoresis analysis. Lane 12 in figure 3 show the size markers of SPP1 digested with EcoR1 while lanes 6 and 7 show samples of pK184hlyA and pBluescript digested with EcoR1 and Pst1. Lane 4 was loaded with plasmid DNA from haemolytic cells digested with EcoR1 and Pst1 while lane 5 was loaded with EcoR1 and Pst1 digested DNA from non-haemolytic cells. There was a lack of technical success in both lanes due to no bands appearing in lane 4 and only a single band appearing in lane 5. Theoretically, two bands should appear in both lanes after successful to allow for fragment identification. A possible explanation for the single, large fragment in lane 5 is that successful digestion did not take place and the plasmid was only cut at one restriction site leaving a large linear fragment of plasmid DNA. The absence of bands in lane 4 could be because there was not enough plasmid loaded into the lane. Another possibility could be that low plasmid yield as obtained when eluting the experimental samples in order to purify it. Lanes 8 and 9 belonged to another group and show technical success as two bands were present in both the haemolytic (lane 8) and non-haemolytic (lane 9) lanes. If the
Charlotte Perkins Gillman and Edgar Allen Poe are both well-known and greatly respected writers in history with similar, but unique writing styles. They both use an unreliable narrator to mislead the reader, but slowly drop hints that something is a little off. In Gilman’s The Yellow Wallpaper she tells a story narrated by a woman in the late 19th century who has been ordered to get as much rest as possible because of her “temporary nervous depression.” As the story progresses, she starts to slowly lose her sanity from being condemned in her room for so long, and eventually develops a scary obsession with the wallpaper. Poe’s short story, the Cask of Amontillado, is narrated by an Italian man named Montresor who has vowed to get revenge for
The short story, " The Yellow Wallpaper", written by Charlotte Gilman, and "The Cask of Amontillado" written by Edgar Allan Poe, are stories in which the plots are very different, but share similar qualities with the elements in the story. "The Cask of Amontillado" is a powerful tale of revenge, in which the narrator of the tale pledges revenge upon Fortunato for an insult. "The Yellow Wallpaper" is a story about a woman, her psychological difficulties and her husband's therapeutic treatment of her illness. She struggles over her illness, and battle's her controlling husband. The settings in both stories are very important, they influence the characters, and help with the development of the plot.
Organic inks, which are my personal professional preference, are the safest on the market. These types of inks are typically derived from plant matter. Vegan inks are also in the same class as the organic inks as well and are also ranked just as safe as organic inks.Organic inks are also safe to digest. I personally have no plans on digesting tattoo ink, but if I planned on doing so at least I know that it is safe.
Setting is a critical part of any story, developing both the time and place in which the story takes place, as well as mood and tone of the text. This certainly takes no exception in Charlotte Gilman’s “The Yellow Wallpaper.” Not only does the setting in “The Yellow Wallpaper” achieve the above, but also it goes one level deeper by giving the reader insight into the narrator’s mindset. By utilizing the setting as described by the narrator, along with the knowledge of the narrator’s battle with hysteria, the reader can fully interpret the setting, its impact on the narrator, as well as determine Gilman’s implications throughout the story.
MacPike, Loralee. "Environment as Psychopathological Symbolism in 'The Yellow Wallpaper.’” Twentieth-Century Literary Criticism, edited by Thomas J. Schoenberg, vol. 201, Gale, 2008. Literature Resource Center, go.galegroup.com.gmclibrary.idm.oclc.org/ps/i.do?p=LitRC&sw=w&u=mill30389&v=2.1&it=r&id=GALE%7CH1420082948&asid=562f132388d74c4bd92439b5842a2fe7. Accessed 25 Oct. 2017.
Construct a manual calibration plot of experimental absorbance versus actual Ca2+ concentration (pink line) using finely lined graph paper. Include proper title and figure #. Label the axes. Using the same graph paper plot experimental absorbance versus experimental Ca2+ concentration (green line). Include a legend to identify both lines
In today’s times, kids all over the world are making different types of slimes to keep themselves entertained. Slime has been around for many years, but recently there has been new ways to make slime at home. Slime can be made using many different ingredients. Some common ingredients used to make slime include; Borax, baking soda, contact lens solution, water, and liquid starch. All of these slime recipes have one ingredient in common, glue. Kids have an array of substances they can add to their slime mixture to create more fun. Additional substances can include food coloring, glitter, Orbeez, small toys, beads, or even confetti. Does decorating slime mixtures increase the consistency of a standard slime mixture? Orbeez can make the slime feel
The band pattern seemed to match the calculated, but have some bands supposedly missing, according to the Table 1 below. On the gel, there is a faint band at ~700 bp, a band at ~2000 bp, a band at ~4500 bp, and a band at ~7000 bp. These bands correspond to #7, #6, #4, and #3, respectively on the left table.
= Before conducting the experiment I would conduct a simple test for the protein by placing a sample of the albumen into a test tube and add biurett reagent. This contains copper (II) sulphate and sodium hydroxide.
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