840:153g:Projects/project26: Difference between revisions
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8.Once then DNA is in our vector we must grow the ''E. coli'' then test with controls. | 8.Once then DNA is in our vector we must grow the ''E. coli'' then test with controls. | ||
9.Testing will be done using SDS PAGE. We will compare the length of the control and our experiment E. coli. If the length of the experiment is longer than the control, then the gene for Atrolysin A is present. | 9.Testing will be done using SDS PAGE. We will compare the length of the control and our experiment E. coli. If the length of the experiment is longer than the control, then the gene for Atrolysin A is present. | ||
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http://scholar.google.com/scholar?hl=en&q=getting+DNA+from+snake+skin&btnG=&as_sdt=1%2C16&as_sdtp http://en.wikipedia.org/wiki/Site-directed_mutagenesis http://www.springerlink.com/content/kw1716u555371764/fulltext.pdf http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 | |||
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Revision as of 13:31, 13 September 2012
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Team Members
The cloning of Atrolysin A from Crotalus atroxWe are going to clone the gene for the the toxin Atrolysin A,Genbank accession number U01234. E. coli will not be effected by the toxin. 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 cut them out by site directed mutagensis. 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. Procedure 1.Make DNA buffer and prepare for the DNA extraction from the snake skin. Buffers needed are Tris/HCl buffer and TE buffer. 2.Use agarose gel electrophoresis to find if the DNA is present in our buffer solution. 3.Once we know the DNA is present we must then PCR the sample to amplify the coding sequence. 4.With the amplified sample we then will find our coding region and sequence it to see if the coding sequence grows from 1260 base pairs. After sequencing we then must look at the sequence to see if we can view what might be introns. 5.If introns are present depending on how many are in the sequence we then must remove them with site directed mutagenesis 6.Once the introns are removed we must then remove the Restriction site PstI with site directed mutagenesis 7.Once we have all the introns and restriction site removed we must then add the biobrick parts and then insert the DNA into our vector. 8.Once then DNA is in our vector we must grow the E. coli then test with controls. 9.Testing will be done using SDS PAGE. We will compare the length of the control and our experiment E. coli. If the length of the experiment is longer than the control, then the gene for Atrolysin A is present. References: http://scholar.google.com/scholar?hl=en&q=getting+DNA+from+snake+skin&btnG=&as_sdt=1%2C16&as_sdtp http://en.wikipedia.org/wiki/Site-directed_mutagenesis http://www.springerlink.com/content/kw1716u555371764/fulltext.pdf http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100
Important Results and Milestones
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