Alibrio Ficheri Lab Report

Superior Essays
A. fischeriThe overall purpose of this study was to create a genomic library of Aliivibrio fischeri (A. fischeri) thus aiding in creating a restriction map of the lux operon. It also employs typical molecular techniques important for biologists to understand. In this portion of the lab, the chromosomal DNA (chDNA) will be isolated. Its purity will be measured using spectrophotometric analysis. Lastly, the DNA will be digested and verified via gel electrophoresis.

A. fischeri is a gram negative rod shaped marine bacteria that exhibits bioluminescence and is both found free-living or in symbiosis. It has a variety of hosts such as the bobtail squid and is named after the German scientist Bernhard Fischer (Garrity, 2005). Bioluminescence in
…show more content…
fischeri chDNA. Lysing the pellet of A. fischeri required the hydrogen bonds, hydrophobic interactions, and Van der Waals forces to be disrupted. This was done by the detergent sodium dodecyl sulfate (SDS), which was present in the re-suspension buffer added and created protein-lipid-detergent complexes. Proteinase K is added to digest proteins and any nucleases that might damage the DNA (Peant and LaPointe, 2004). RNase is added to avoid impurities from RNA. The addition of the chaotropic salt guanidinium chloride present in the lysis buffer helps the DNA adhere to the silica surface of the DNeasy Mini spin column by forming a cationic bridge that connects the DNA and silica gel (Yang et. al., 1998). It also denatures lipids and proteins, preventing them from adhering by weakening their hydrophobic interactions, causing them to be simply washed away. Ethanol is added to further ease DNA binding while continuing to eliminate the lipids and proteins by creating a hydrophobic environment. Wash buffer 1 washes away proteins while Wash buffer 2 is used to remove salts. DNA grade water is added in the end allow the DNA to renature and detach itself from the silica spin column (Qiagen,

Related Documents

  • Improved Essays

    Then the Alkaline Protease functioned as the inhibitor of any DNases and denatured dsDNA to ssDNA shortly, by disturbing H-bonds between bases. Finally, the Neutralizing Solution was helpful into stabilizing the pH, from a basic solution formed by the lysis and protease solution. The DNA was…

    • 1208 Words
    • 5 Pages
    Improved Essays
  • Superior Essays

    Gram Catalase Test Report

    • 1163 Words
    • 5 Pages

    The reasons range from diagnosing a disease in a patient, so as to know how it can be handled and treated, to knowing the appropriate microorganism to be used for making certain foods or antibiotics. Also, it can lead to many new discoveries such as new species or evolution of new species. This study is done by applying all of the methods and techniques that have been learned and used in the microbiology laboratory for the identification of the unknown bacteria’s. In this experiment, multiple of tests were conducted to provide the fermentation abilities, existence of specific enzymes, and certain biochemical reactions. Observations obtained from the tests were compared to the unknown bacteria identification key help with the identification process.…

    • 1163 Words
    • 5 Pages
    Superior Essays
  • Improved Essays

    The role of the EDTA in this solution is to bind to the cations in the lipid bilayer, which weakens the cell’s envelope. At this rate, the EDTA limits the DNA from degradation. After adding this solutions, the group resuspend each tube by vortexing each tube. Vortexer is a machine that mixes small samples of liquids. Adjusting the pipette to 200 µl, we add the second Miniprep solution or MP 2.…

    • 1324 Words
    • 5 Pages
    Improved Essays
  • Improved Essays

    Step 5. The AmBic buffer was first added. Then, the DTT was added to the obtained affinity resin slurry in order to reduce the disulfide bonds of the protein. The mixture was incubated. Step 6.…

    • 824 Words
    • 4 Pages
    Improved Essays
  • Great Essays

    Mannitol Salt Agar

    • 1936 Words
    • 8 Pages

    The first test involved the Mannitol Salt Agar (MSA). MSA is an example of both a selective and a differential media. It selects for halotolerant bacteria and with the aid of phenol-red differentiates for the ability of a microbe to ferment mannitol or not. If a microbe has the ability to ferment mannitol, it produces acidic waste products which react with the phenol-red to yield a yellow color. If the microbe is unable to ferment mannitol, it will consume the proteins present and will produce a pink/red color.…

    • 1936 Words
    • 8 Pages
    Great Essays
  • Improved Essays

    Unknown Lab Report

    • 1511 Words
    • 6 Pages

    Determining the cell shapes will help with the process of elimination organisms. Under microscopic examination for my unknown 16 broth, it appears to be a coccus shape and a gram positive. Gram staining can determine cell morphology, size, and arrangement. In a positive gram stain, a primary stain called crystal violet is used to stain the cell. For every new stain, mordant, decolorizing agent, and counterstain, it must be rinsed with distilled water.…

    • 1511 Words
    • 6 Pages
    Improved Essays
  • Improved Essays

    The16S rRNA primers used in this PCR, are complimentary to sequences conserved in all bacteria and will target sites of variation. These sites of variation were the regions of the DNA amplified by PCR. The products of PCR cannot be sequenced correctly without purification. Purification removes left over reactants and enzymes. A spin column-collection tube complex is used to collect the DNA during a series of centrifugation techniques with different buffers.…

    • 1466 Words
    • 6 Pages
    Improved Essays
  • Improved Essays

    Transformation allows the bacteria to be introduced to a foreign plasmid. The bacteria is then amplified by the plasmid, allowing for larger quantities of it to exist. Plasmids are small circular pieces of DNA that contain genetic information that may enhance the growth of bacteria. The information is a protein, that when encoded, will make the bacteria, in this E. coli, resistant to any type of antibiotic. Plasmids came about as a result of bacteria evolving.…

    • 722 Words
    • 3 Pages
    Improved Essays
  • Superior Essays

    Enzymatic Assay Lab Report

    • 2335 Words
    • 10 Pages

    The periplasm can be isolated by spheroblasting, then centrifugation (Thein et al., 2010). To do this, the pellet was re-suspended in a solution containing Ethylenediaminetetraacetic acid (EDTA), tris buffer, lysozyme, and sucrose. Inner membranes of Gram-negative bacteria can be uncovered by using a mixture of lysozyme and EDTA, which causes the outer membrane to break down (Thein et al., 2010), liberating the periplasmic contents. The sucrose was used to maintain an isotonic solution to prevent lysis of the sphaeroplasts, which are simply cytoplasmic membrane bound cells. The sphaeroplasts were then spun down using centrifugation, and the supernatant was removed, which presumably contained the contents of the periplasm.…

    • 2335 Words
    • 10 Pages
    Superior Essays
  • Superior Essays

    Escherichia Coli

    • 819 Words
    • 4 Pages

    The procedure uses different buffers to optimize the lysis procedure making sure just DNA is absorbed while undesired products are not retained and in opposite are found in the flow-through. These buffers are: P1 (a resuspension buffer that hydrolyzes not DNA but RNA since it contains the RNase enzyme), P2 (a lysis buffer, it opens, breaks or lyse the cell wall and contains SDS and NaOH), and N3 (a neutralization buffer that contains an acetate buffer which helps in the neutralization of NaOH and guanidine hydrochloride, which protagonist the denaturation of proteins, as well N3 aim in the DNA attachment to the gel membrane in the column due to its high concentration of salt). During the washing and elution processes, Buffer PB is used for a brief wash step and in this way efficiently get ride of the endonucleases and guarantee a no degraded DNA. Buffer PE is also used to wash and efficiently remove salts, highly efficient buffer for DNA cleanup procedures. It is composed of 10 mM Tris-HCl pH 7.5, 80% ethanol.…

    • 819 Words
    • 4 Pages
    Superior Essays