So two of my sisters have asked for an explanation of the big blue assay that I am working on. I've decided to break it up into 3 parts. This assay is used to determine genetic changes in transgenic rats (you can also use mice or cells) following exposure to a substance. The rats have the LacI and LacZ gene incorporated on the 4th chromosomes which allows for you to determine genetic changes. The LacI is a genetic region that is control the ability of a cell to breakdown lactose, its acts as a repressor. If there is a change sequence in the area of the LacI (a region of about 1300 bp in length) it will generate beta-galactosidase. Beta-galactosidase will breakdown a substrate called X-gal which turns blue. And this is why it is called the Big Blue Assay. Although the company Stratagene has also created the same assay using a temperature sensitive based mutations but still gets called the big blue assay. The tissue that I am using is from a 28- repeat dose exposure study using PCB 3 and a PCB metabolite. I am using the lung from the male exposure for my particular experiment although last year I was helping Jim do the big blue using the liver from female exposure group. So the first thing that I do is isolate the DNA from the tissue. This involves pulverizing the tissue then adding enzymes that will digest the proteins, lipids, and RNA. Then I apply the DNA to a dialysis cup that will allow for small molecules to be separated from the DNA (the dialysis cup sits on the surface of buffer). The DNA will dialyze for about 48 hours and as a result will be very viscous. And then I use this DNA for the next step.
"How embarrassing! I thought I had removed the laces from this shoes." - Bill Dauterive
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