Difference between revisions of "Team:Tufts/Results"

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The presence of 4kb bands confirms the strong presence of Cas9 in at least some of the samples.
 
The presence of 4kb bands confirms the strong presence of Cas9 in at least some of the samples.
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The gel of samples 1-5.A faint band (not clearly visible in image) at ~4.2 kb in 3, indicates Cas9 presence/success.
 
The gel of samples 1-5.A faint band (not clearly visible in image) at ~4.2 kb in 3, indicates Cas9 presence/success.
  
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Revision as of 23:39, 18 September 2015

The first few steps of the project involved purifying and checking both of the genes for Cas9 and aTcdB. This involved purifying the products of PCR and running them on electrophoresis gels, as well as using a nanodrop machine to identify the concentration and purity of DNA recovered.

  1. pHis1522-aTcdB
Sample Concentration (ng/μL)
A 135.5
B 170.2
2. pHsp70-Cas9
Sample Concentration (ng/μL)
A 32.9
B 33.3
C 118.3
The presence of 4kb bands confirms the strong presence of Cas9 in at least some of the samples. The genes were then ligated into vectors and transformed into JM-109 E. Coli bacteria cells in order to replicate the vectors. The bacteria were left to grow overnight, and then the plasmid was isolated from them. pHsp70-Cas9
Sample Concentration (ng/μL)
A 45.7
B 46.3
C 40.4
Finally, the Cas9 gene was digested, purified, and ligated upstream of the aTcdB gene in the pHis1522 plasmid. The results were confirmed on a protein gel. NanoDrop of pHis1522-aTcdB-Cas9 Results
Sample Concentration (ng/μL)
1 49.9
2 72.1
3 1702.6
4 426.6
5 42.7
The gel of samples 1-5.A faint band (not clearly visible in image) at ~4.2 kb in 3, indicates Cas9 presence/success.
In addition to the creation of the Cas9-aTcdB protein, the lab also worked on the creation of a template for the easy customization of the gRNA in the CRISPR for Cas9. The sequence for the gene was ordered, then underwent PCR when it arrived. The PCR was confirmed to have worked by Gel electrophoresis. Unfortunately, an attempt to ligate it into the pHis1522 plasmid failed, confirmed by means of DNA sequencing. No more attempts could be made by the time of the wiki freeze, but work on all parts of the project is still ongoing.