840:153g:Projects/project17/2011/11/17: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
(Autocreate 2011/11/17 Entry for 840:153g:Projects/project17)
 
No edit summary
 
Line 1: Line 1:
Originally, we wanted to clone the gene responsible for making the monarch butterfly taste bad to birds and/or make them sick.  We were unable to find any genes responsible for this, since they actually sequester normally toxic chemicals from the milkweed plant.  The plants produce these chemicals (known as cardenolides) via a biochemical pathway of which the enzyme progesterone 5-beta reductase is a part. This enzyme metabolizes progesterone into 5-beta-pregnane-3,20-dione, which is a steroid metabolite which we will test for using high performance liquid chromatography. Since there is no recorded gene sequence for this enzyme in the milkweed plant, we will be using a gene from a species of spike moss. We plan to order our Selaginella moellendorffii online from Plant Delights Nursery.  Our gene sequence was located in the NCBI GenBank. Its accession number is NW_003314261. It is 1185 base pairs in length, and contains no introns.
Today we ran our purification products on a gel with two different ladder amounts so that we could determine the concentrations of our gene and plasmids in order to set up ligation samples with known ratios of vector and insert.  Our gene did not show up on the gel, and looking back in our lab notebook showed us that the PCR using the primers with BioBrick extensions was probably not successful because the gel we ran for that was inconclusive (but we went ahead with the digestion, ligation, and transformation anyway). Our gel also showed two bands for our 4120 plasmid, which means it was not fully digested. After break, we plan on redoing PCR with the BioBrick extension primers and run a gel on it to see if we can get our gene with BioBrick extensions this time (which we then can digest in order to ligate into our plasmids that were fully digested).
 
Reference article: Evolution of steroid-5-alpha-reductases in comparison of their function with 5ß-reductase
DOI: 10.1016/j.ygcen.2009.08.004

Latest revision as of 15:53, 17 November 2011

Today we ran our purification products on a gel with two different ladder amounts so that we could determine the concentrations of our gene and plasmids in order to set up ligation samples with known ratios of vector and insert. Our gene did not show up on the gel, and looking back in our lab notebook showed us that the PCR using the primers with BioBrick extensions was probably not successful because the gel we ran for that was inconclusive (but we went ahead with the digestion, ligation, and transformation anyway). Our gel also showed two bands for our 4120 plasmid, which means it was not fully digested. After break, we plan on redoing PCR with the BioBrick extension primers and run a gel on it to see if we can get our gene with BioBrick extensions this time (which we then can digest in order to ligate into our plasmids that were fully digested).

Retrieved from "https://openwetware.org/mediawiki/index.php?title=840:153g:Projects/project17/2011/11/17&oldid=567189"