Testing of Intercellular Material for DNA through Agarose Electrophoresis
Purpose: The point of this lab was to determine whether or not DNA was actually extracted in the prior week’s experiment, in which E. Coli bacteria’s was lysed and through a series of chemical extractions it’s inner contents were harvested.
Methods: 4.5 mL E.Coli EDTA suspension was pipetted into a conical tube. After this 0,25 mL lysosome solution was put inside the same tube. Both were incubated at 37°C for so minutes. Once out of the incubator, 0.5 mL of 10% SDS was added. In order to ensure a good mixing of the liquids the tube was inverted continuously for several minutes. Incubation occurred once more but this time at 60°C for 10 minutes. Once cold down to room
It was loaded into the gel instead of in the well leading to no results. There should have been a series of bands similar to well three. In lanes three, 5, and 6 we see the bands labeled A,B and C. Due to the short distance traveled down can discern that these bands are in fact DNA as it is both bulky and negatively charged. In lane seven one can see the band in the beginning, which is identified, as tRNA. E and F represent the left of “junk” from the inside of the cell that made it’s way into the sample such as mRNA and proteins. This can be told by seeing that the small materials traveled very far down the gel and were not removed by DNase. What truly tells one whether or not he or she extracted DNA are the blank spots on the gel. In lane four and eight there are missing bands. This is due to the fact in these samples enzymes where added to break down the nucleic acids, DNase in the case of lane four and RNase in eight, thus causing a gap where they should appear. The data that was collected seems to indicate that the sample that was extracted was done properly and yielded DNA. It should be noted that the lanes three and four were switched when adding the material into
The unknown bacterium that was handed out by the professor labeled “E19” was an irregular and raised shaped bacteria with a smooth texture and it had a white creamy color. The slant growth pattern was filiform and there was a turbid growth in the broth. After all the tests were complete and the results were compared the unknown bacterium was defined as Shigella sonnei. The results that narrowed it down the most were the gram stain, the lactose fermentation test, the citrate utilization test and the indole test. The results for each of the tests performed are listed in Table 1.1 below.
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
The two modes of analysis that will be used to identify an unknown insert piece of DNA would be plating the transformation cells onto LA plates that have either ampicillin or chloramphenicol and PCR. We will use the PCR thermocycler to denature the restriction enzymes that were specifically used to assimilate the vector DNA. It is important to use the PCR thermocycler because denaturation of the restriction enzyme will prevent the restriction enzyme from cutting the vector DNA, after the insert DNA has assimilated to the vector DNA. After the addition of specific primers that complement the base pair to its corresponding target strand, PCR will be used. Subsequently, Taq polymerase will be used to determine whether the insert DNA has been properly assimilated to the vector DNA. Within this specific situation, the target strand will be the insert DNA. After we let the PCR thermocycler run for approximately 2 ½ hours, we will then put our PCR products in the gel and run the gel to completion. After the gel has run to completion, we will then take a photograph of the gel using the UV transilluminator with the assistance of our TA. If the insert DNA was properly assimilated to the vector DNA, then our corresponding gel photo would have one band. After the cells have been transformed, we would g...
Living organisms undergo chemical reactions with the help of unique proteins known as enzymes. Enzymes significantly assist in these processes by accelerating the rate of reaction in order to maintain life in the organism. Without enzymes, an organism would not be able to survive as long, because its chemical reactions would be too slow to prolong life. The properties and functions of enzymes during chemical reactions can help analyze the activity of the specific enzyme catalase, which can be found in bovine liver and yeast. Our hypothesis regarding enzyme activity is that the aspects of biology and environmental factors contribute to the different enzyme activities between bovine liver and yeast.
Upon completion of the experiment we were able to examine the DNA. First, the electrophorese
The given DNA ladder sample and each individual ligation samples were run on 40ml of 0.8% agarose in 1x TAE buffer for approximately sixty minutes at 110V. The appropriate volume of 6x GelRed track dye was used after it was diluted to a final concentration of 1x and incubated for thirty minutes. Finally, the gel was illuminated under UV light and analyzed.
Introduction: Gel electrophoresis is used to separate molecules like RNA, DNA and proteins. DNA fragments are separated by size and proteins are separated according to the size and their charge. Gel electrophoresis use positive and negative electrode to separate charge particles. The charged particles are attracted to opposite charges (Purdue University 2012). As the DNA has a negative charge the electrical circuit which has positive charge is placed at the bottom of the gel. This causes the smaller DNA particles to move quickly through the gel (Brooker 2012).
Paabo’s team, from Leipzig, Germany, used a method of amino acid content as a way of measuring extractible DNA from the bones. The amino acid method was a...
Then, using a fresh tip each sample, I transferred of the enzyme to each separate tube of the DNA samples. By adding the enzymes, this will cut the DNA molecules into small pieces when we place it into the gel and let it
LAB REPORT 1st Experiment done in class Introduction: Agarose gel electrophoresis separates molecules by their size, shape, and charge. Biomolecules such as DNA, RNA and proteins, are some examples. Buffered samples such as glycerol and glucose are loaded into a gel. An electrical current is placed across the gel.
Meaning for 50ml, 0.4 grams of agarose was weighed on a scale. And with a 250ml flask, 50ml of 1X TBE buffer was added as well as the 0.4 grams of agarose (Upadhyaya, 2017). Then plastic was placed over the flask and put in the microwave for one minute. After safely retrieving the hot flask from the microwave with a mitten, it was observed to ensure that the agarose was completely dissolved and was placed on a table to cool down to not burn the casting tray; once cooled 2.5µL of ethidium bromide was added to the agarose solution, which was used to illuminate the gel once placed under UV light (Upadhyaya, 2017). Before pouring the solution into the casting tray, a comb was placed on the middle of the edge of the tray to create wells once the gel was solid, allowing for multiple DNA samples to be used at the same time to be compared against each other. After the gel was solidified, the tray was repositioned as the positive pole was closer to the wells which then would not show much results. After adding 250ml of 1X TBE buffer to the tray, an almost equal amount on both sides, submerging the gel, the comb was removed and the wells were present (Upadhyaya,
There are other ways to extract DNA. You can even do it at home with in house items. You can even do this easy experiment in three easy steps. Blend a bag of peas then strain them into a blender. After pouring all that into a cup then poor liquid detergent into the
== § Test tubes X 11 § 0.10 molar dm -3 Copper (II) Sulphate solution § distilled water § egg albumen from 3 eggs. § Syringe X 12 § colorimeter § tripod § 100ml beaker § Bunsen burner § test tube holder § safety glasses § gloves § test tube pen § test tube method = == = =
The liquids used were distilled water, and a starch solution, in line with the guiding question. Dialysis tubing was used because it performed similarly to an actual cell while being visible to the naked eye. First, I soaked four strips of dialysis tubing in water for 5 minutes, afterwards I knotted off one of the ends for all of the tubing. After, I filled two with a half tablespoon of distilled water, and another two with half a tablespoon of the starch solution. Next I measured the lengths, widths, and weights of the cells. I proceeded to leave the four cells to soak in distilled water for about 24 hours. After leaving them in distilled water for about a day, I extracted the cells from the distilled water and remeasured the measurements mentioned before. In hindsight, this was an ideal method to investigate the guiding question because the dialysis tubing functions almost identically to a living cell while being easier to observe and handle at the
doubt be linked to the scene. All that is needed to extract DNA is one