Electrophoresis Gel Case Study Answers

The following tests according to the lab manual were performed: gram stain, fermentation tubes, methyl red, vogues proskauer, sulfur, indole, motility and growing it up on MacConkey agar. The gram stain was performed incorrectly the first time. This is because the decolorizer was not on the bacterium slide for long enough, giving a false outcome.

We use eight lanes putting the enzymes, and the DNA of the suspects to the lane. It is important to do this slowly and with care so as to ensure we put each solution in the correct tubes. You also have to plug it to the power source. Red is the negative while black is the positive and it should be at 150V for about forty minutes. Evaluation of the DNA bands

Therefore the ratio of A260/230 is the concentration of DNA/ the concentration of other containments (Wilfinger). From these values one can determine that the sequencing process was successful. These values displayed that the sequencing reaction worked, but did not give the desired results and did not have a concentrated product. The amount of the DNA sample and water had to be adjusted in the tube that was going to be sent to the Cornell lab. The amount that was supposed to be used was 1.5ul of mtDNA. This value was determined by 100ng of DNA required x volume of DNA in ul divided by the concentration of DNA. The instructor simply advised to double the amount of mtDNA that would be placed in the tube. The volume of DNA used was 3uL, and the volume of sterile water used was 13.4uL. The volume of the primer used was 1.6uL(which was not adjusted). Figure 2 displays the reverse complement of the mtDNA that was obtained from the Cornell lab sequencing the mtDNA sent to them. This was then used to compare it with the CRS mtDNA. Figure 3 displays the aliment of the CRS and the revers compliemt of the mtDNA sent to the Cornell

21. The -DNA/LB plate had an even lawn of bacteria and looked off-white while the +DNA/LB/AMP plate had no bacterial growth.

My partner and I choose glucose, colchicine, cold temperature (0 degrees Celsius), and warm temperature (37 degrees Celsius) as the addition factors. The tetrahymena were exposed exposed to their condition for ten minutes before the India ink was placed in their microcentrifuge. The tertrahymena were fixed on a slide every ten minutes after the ink was placed in the microcentrifuge. In this experiment there are two controls. There is positive control and a negative control. The untreated sample is tetrahymena that did not have any other conditions besides the ink. Then before the sample was placed on the slide they were fixed. The treated sample was tetrahymen mixed with

The test tube was labeled with the bacteria identifying number. The cap on test tube was removed, and the lip of the test tube was flamed. Next, the Bunsen burner was used to sterilize the inoculating loop. Then, bacteria were picked up from the working plate with the loop and the agar was inoculated. The loop was then re-flamed. Finally, the plate was placed in the 37˚C incubator and left to sit for 48 hours and any changes in color were observed. A negative result appears green, and a positive result appears blue. This is because it tests for the organism’s ability to use citrate as its sole source of carbon, and if it does then it produces ammonia and ammonium hydroxide which make the medium basic, changing the green agar blue (Leboffe & Pierce,

I believe the bacteria in tube 30A is E. faecalis, and the bacteria in tube 29B is E. coli. For tube A, the DNase test yielded a negative result, with absolutely no clearing on the agar. Only three bacteria would produce a negative result: S. epidermidis, E. faecalis, and M. luteus. From there, I performed a catalase test, in hopes of attaining a negative result. The result of the catalase test was, in fact, negative. This eliminated both S. epidermidis and M. luteus because these bacteria would have yielded a positive result. The result of the catalase test was completely negative, as evidence by no bubbles being produced after the addition of a drop of hydrogen peroxide. For tube B, the first test I performed was a EMB plate test. The result

coli are picked and the plasmids are purified. The purified plasmids are used as a template for the sequencing reaction. The objective of the lab was to learn how to use the polymerase chain reaction (PCR) to amplify the small subunit ribosomal RNA gene from a bacteria colony, also be able to run an agarose gel to visualize the resulting PCR amplifications and extract the amplified DNA from the agarose gel.

One MR-VP broth was the control tube, which was not inoculated at all. The other MR-VP broth was the treatment broth that was inoculated with the colony 2 stock culture at 30 degrees Celsius for five days. The reason for completing this test first after gram staining was because it was a 5-day test that could be left over the weekend. There were no variables, it was either negative or positive, and there would be no misleading guesses. The plan I had going into this was to perform all the tests possible with no variables or not determined results to reduce the potential for errors. On Monday I will get the control and treatment inoculated test tubes back to determine wither the unknown organism is positive or negative based off of the color in which the broth will

The DNA extraction results, along with the PCR product, did not fare well. There was not enough product produced to be viable in the later stages of the experiment, so a backup was used in place of the original product.

When picking up the gel, the gel broke in half. This could have altered the results because some of our DNA could have broken off. Although it appeared that we saved the DNA, there is a slight chance that some of the DNA could have broken off. Also, my assistants and I had difficulty measuring the agarose powder. We might have poured too much of the powder, which could have made the gel too thick. Next time, we will be more cautious when measuring the powder to have more accurate results.

The Eppendorf tube was then placed within a centrifuge for 10 minutes to separate the insoluble DNA from the rest of the solution. When the 10 minutes were up, a DNA “pellet” appeared at the bottom of the Eppendorf tube and a pipet was used to decant the solution. Once as much liquid was removed from the Eppendorf tube as possible, 1 mL of 70% ethanol was added to the tube, and it was inverted another 5 times. The sample was then placed within the centrifuge for another 5 minutes at maximum speed. The DNA pellet should still be visible within the Eppendorf tube and using a pipet, the ethanol was decanted from the Eppendorf tube.

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)

PCR (Polymerase Chain Reaction) is a laboratory technique that takes specific fragments of DNA and amplifies them using primers and a polymerase that can withstand high temperatures. The materials needed to complete this experiment include the DNA fragments that are to be amplified, two primers (one to attach to the top strand of the DNA fragment of interest and one to attach to the bottom strand on the other end), Taq DNA polymerase, dNTP mix, MgCl2, PCR buffer and the PCR machine. Primers are necessary for the Taq DNA polymerase to attach to in order to begin copying because primers can only attach to an existing piece of DNA. Taq DNA polymerase is necessary for PCR rather than DNA polymerase found in our bodies because when the DNA fragments of interest are heated in order to denature them and separate the two complementary strands, DNA polymerase also denatures. Taq DNA polymerase is taken from a strain of bacteria (Thermus aquaticus) that is taken from the hot springs of Yellowstone National Park, and it can withstand boiling temperatures which makes it effective for PCR. dNTP mix is a mixture of nucletotides that the polymerase uses to create the new

The E. Coli DNA Isolation and Analysis lab’s purpose was to isolate E. Coli DNA and analyze it using gel electrophoresis. We were able isolate E. Coli DNA and we could see the bacteria growing on our plates. Because of the way we streaked it across, We were able to see the long lines of the organisms, but also smaller colonies as the number dwindled on later sections of the plate. The salmon sperm DNA we gathered made a long, bright strip during gel electrophoresis, compared to the E. Coli, which made a single band. This means that the salmon sperm DNA had a large variation of lengths in its strands, whereas the E. Coli DNA had a set length. Another thing is that the salmon sperm DNA lanes had a much brighter glow, while the E. Coli had a much dimmer glow. This signifies that the salmon sperm had much more DNA than the E. Coli cells. The E. Coli bands were on the same level on the ladder as the salmon sperm DNA’s bulk, showing they had mostly the same