Difference between revisions of "840:153g:Projects/project26"
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The cloning of Atrolysin A from Crotalus atrox
We are going to clone the gene for the toxin Atrolysin A,Genbank accession number U01234. The toxin is produced by Crotalus atrox, which is the Western Diamondback. The E. coli that we will use as a vector will not be effected by the toxin if produced. The Atrolysin toxin affects platelet adhesion inside the body. The toxin works best at a pH of 8.0 and denatures in an acidic environment. This is how the snake can eat its prey and not be affected by the venom. The Atrolysin A is only found in mRNA sequence so we have to isolate the coding sequence and amplify it with PCR. Once we have the gene isolated we need to amplify it and see if the coding sequence grows from its initial 1260 base pairs. If it does this means introns are in the sequence. If introns are involved we then must remove them. Removing them by splicing and PCR. We have also discovered a restriction site in our gene and it has to be removed by site directed mutagenesis. If we are able to isolate and clone the coding sequence, we will then test the protein on SDS gels against a control E. coli to see if the protein is present. Our primers we are using are Forward: 5’ ATG GAA AGA CTC ACC AAA AGA TAT GTT GAC CTT GTC ATA GTT G 3’. The Reverse Primers: 5’ TCA AAT CTG AGA GAA GCC AGA GGT TGA TTT GTA GGC TGT A 3'
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"Agarose gel electrophoresis." OpenWetWare. October 2012. http://openwetware.org/wiki/Agarose_gel_electrophoresis.
Anderson, John. Part:BBa_I0500. 4 Aug. 2006. Registry of Standard Biological Parts. 4 Aug. 2006. http://partsregistry.org/wiki/index.php?title=Part:BBa_I0500.
"Crotalus atrox hemorrhagic toxin a, atrolysin a (Ht-a) mRNA, partial cds." National Center for Biotechnology Information. 2012. http://www.ncbi.nlm.nih.gov/nuccore/U01234.1.
Eguchi, Tomoko and Yukinori. High yield DNA extraction from the snake cast-off skin or bird feathers. 19 May 2000. University of Ryukyus. 19 May 2000. http://scholar.google.com/scholar?hl=en&q=getting+DNA+from+snake+skin&btnG=&as_sdt=1%2C16&as_sdtp.
Fetzner, James W. Extracting High-Quality DNA from Shed Reptile Skins: A Simplified Method. June 1999. Brigham Young University. June 1999. http://www.biotechniques.com/multimedia/archive/00014/99266bm09_14694a.pdf.
Site-Directed Mutagenesis. 22 Aug. 2012. Wikipedia. 22 Aug. 2012. http://en.wikipedia.org/wiki/Site-directed_mutagenesis.
"Standard PCR Setup." Openwetware. October 2012. http://openwetware.org/wiki/840:153g:Materials.
Important Results and Milestones
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