Aliivibrio Fischeri Lab Report

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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
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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, …show more content…
With this spectrum, the purity and concentration of the chDNA can be determined. RNA and DNA have a maximal absorbance at 260 nm while proteins have a maximal absorbance at 280 nm (Winfrey et al., 1997). The absorbance values are converted into concentrations using the Beer-Lambert law A = εcL, where A is the absorbance at 260 nm, ε is the molar absorptivity of dsDNA (0.02 μg/ml-cm), c is the concentration, and L is the path length of the cuvette (Teare et al., 1997). The A260/280 ratio signifies the level of protein contamination; ideally, the ratio should be between 1.8 and 1.9, denoting the relative absence of protein contamination. Because DNA does not absorb light at 320 nm, a pure sample of DNA should have an absorbance at 320 nm that is less than 5% of its absorbance at 260 nm. 230 nm is the minimum absorbance of DNA; a high value at 230 nm indicates contamination with aromatics and salts (Arruda et al., 2013). The 260/230 ratio should ideally be between 1.8 and 2.2. A result below 1.8 would indicate contamination and a larger than normal 230 nm reading. (Winfrey et al.,

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