840:153g:Projects/project17/2011/10/11: Difference between revisions

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(Autocreate 2011/10/11 Entry for 840:153g:Projects/project17)
 
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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.
Our goals for today were to run an RE Digest on our isolated plasmid containing our promoter parts and then run a gel to check that everything was working and the isolation went well, and to set up PCR for our plant DNA.  Using the four combinations of BioBrick restriction enzymes, we created four master mixes and then split each master mix into four parts (three for each of our promoter parts and one with a standard plasmid control). We ran this on the gel, but no Ethidium Bromide was added to the gel, so we will redo this on Thursday. We set up the PCR for three different temperatures using our normal reverse primer (without BioBrick extensions) and our one forward primer. We also made a negative control and two positive controls (using the standard plasmid).  Our goals for Thursday are to redo the RE Digest and and run the plasmids with our promoter parts, as well as our PCR products on a gel to determine what procedures and temperatures worked.
 
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 15:07, 11 October 2011

Our goals for today were to run an RE Digest on our isolated plasmid containing our promoter parts and then run a gel to check that everything was working and the isolation went well, and to set up PCR for our plant DNA. Using the four combinations of BioBrick restriction enzymes, we created four master mixes and then split each master mix into four parts (three for each of our promoter parts and one with a standard plasmid control). We ran this on the gel, but no Ethidium Bromide was added to the gel, so we will redo this on Thursday. We set up the PCR for three different temperatures using our normal reverse primer (without BioBrick extensions) and our one forward primer. We also made a negative control and two positive controls (using the standard plasmid). Our goals for Thursday are to redo the RE Digest and and run the plasmids with our promoter parts, as well as our PCR products on a gel to determine what procedures and temperatures worked.

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