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Revision as of 00:44, 13 October 2016
Experiments
High-Throughput Sequencing
To extract environmental DNA from fecal samples. This extraction will purify genomic DNA from prokaryotic and eukaryotic cells found in the fecal sample.
Concentration protocol to concentrate DNA isolating using the PowerFecal Extraction kit.
Illumina MiSeq Sequencing was used to identify the sequences of the 16S rRNA genes in the fecal samples. This step was undertaking by the Integrated Microbiome Resource at Dal. There protocol was provided to us.
Metagenomic sequencing is also done by the Integrated Microbiome Resource at Dalhousie. It also uses illumina miseq. Their protocol has been provided to us.
Isolation of Bacteria
Congo Red Cellulose media can be used to isolate bacteria that degrade cellulose. It does this because the only carbon source in the media is cellulose. It uses Whatman Paper and gelatin.
Colony PCR has many applications including; verifying that an insert has been successfully incorporated in plasmid constructs, insert orientation, and species determination of unknown samples. The later was the reason for our use of Colony PCR. After streaking fecal sample onto cellulose plates, we picked colonies of different morphologies to find out which species or genus they were. We did this by using primers that targeted conserved sequence found in bacteria so we could amplify the DNA of almost all bacteria.
Instead of PCR clean up, we used ExoSap It. ExoSap It allows for an enzymatic clean-up of left over dNTPs and single stranded DNA. It is a very quick procedure as the only activation ExoSap It needs is a 15 minute 37°C incubation and a 15 minute 80 °C inactivation.
Metagenomic Library
Protocol that takes extracted fecal DNA, prepares it for ligation and clones it into a cosmid. This cosmid can be packaged into phage particles and those can be used to place the cosmid into E. coli cells which will express environmental DNA that had been placed into the cosmid.
Chemical Analysis
Steam Distillation was used to separate the terpenes from pine tree oleoresin without degrading them. Through GC/MS analysis we confirmed that this procedure worked.
These experiments were used to test E. coli and S. cerevisiae tolerance to terpenes, both in pure form and the terpenes isolated from oleoresin.
