Dneasy Extraction Kit

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One of the primary goals of this experiment is to generate a genomic library for A. fischeri, and the first step to do this involves isolating and analyzing the chDNA of the bacteria. This was completed using the Qiagen DNeasy Extraction Kit, which involves a variety of steps including: lysis of the chDNA, the binding of the sample to silica, multiple washings of the sample, and an elution to obtain pure chDNA for analysis (Qiagen, 2006). To start off, 180 μl’s of resuspension buffer was added to the A. fischeri pellet in a 1.5 ml microcentrifuge tube (Qiagen, 2006). In order to deactivate nucleases (so that nucleic acids can be released), 20 μl of proteinase K was added (Qiagen, 2006). After vortexing the sample in order to mix everything…show more content…
Special care was taken to ensure that the spin column did not come in contact with the flow-through. Since the chDNA is bound to the silica at this point, they must be separated using two wash buffers. The initial 500 μl of buffer (AW1: Wash Buffer 1) was added in order to remove contaminants and proteins that could be in the sample using chaotropic salts (Qiagen, 2006). After centrifuging the sample for 60 seconds at maximum speed, 500 μl of Wash Buffer 2 (AW2) was added to remove the salts (this contained ethanol). After centrifuging the mixture for 3 minutes at maximum speed, AW2 was added again and centrifuged (Qiagen, 2006). Once this final centrifuge was finished, the sample was air-dried for 10 minutes at room temperature. Next, a microcentrifuge tube was added beneath the spin column, and 75 μl of DNA grade water was added to the spin column. This was done to rehydrate the DNA and separate it from the silica (Qiagen, 2006). The tube was then incubated for 2 minutes at room temperature and centrifuged at maximum speed for 2 minutes. This step was repeated in order to elute the sample (total volume 150 μl).
Once the elution had been completed, the sample obtained was further analyzed using a Nanodrop 2000 Spectrophotometer. The purity and concentration of the
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