User:Katelyn R. Porter/Notebook/0721 T4 DNA PCR Sequencing

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PCR Sequencing

Purpose: To identify bacteria using polymerase chain reaction (PCR) sequencing.


Materials and Methods

1. A single colony was placed in a sterile tube with 100 µl of water, then incubated at 100º C for 10 min

2. The boiled samples were then centrifuged for 5 minutes at 13,400 rpm

3. 20 µl of a primer/water mister was added to a 16s PCR reaction tube, then 5 µl of supernatant from the boiled centrifuged samples were transferred to the PCR reaction tubes and placed in the PCR machine

PCR Sequence Result.JPG


Data and Results

The sequencing of our bacteria was not successful, resulting in inconclusive data. However, an individual from a previous section, Abigail Fogg, who observed the same transect was able to run a successful PCR sequence and identify two bacteria. After running them through NCBI blast, they were identified as such:

Bacteria PCR Table.png


Chryseobacterium are gram-negative bacilli that are found primarily in soil and water. They can survive in chlorine treated water supplies, indicating some antibacterial resistance. This is congruent with one of our bacterial colonies, which was found in soil, was gram negative, and was somewhat tetracycline resistant. Chryseobacterium also grows in yellow colonies, which was also characteristic of the bacterium we isolated.


References:

1. Kirby, J. Antimicrobial Susceptibility and Epidemiology of a Worldwide Collection of Chryseobacterium spp.: Report from the SENTRY Antimicrobial Surveillance Program (1997-2001). Journal of Clinical Microbiology, 42, 445. Retrieved July 24, 2014, from http://jcm.asm.org/content/42/1/445.full.pdf

2. http://openwetware.org/wiki/User:Abigail_M._Fogg/Notebook/Biology_210_at_AU

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