Lab 4 Transgenic Plants Gene Expression (1)

Lab 4 Transgenic Plants Gene Expression (1) - Transgenic...

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Transgenic Plants/Gene Expression Zachary Bergman Partner: Anuj Shah 3/8/09
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Introduction The transfer of genetic information from DNA to a functioning protein uses multiple forms of RNA to transcript and translate the gene sequence. This lab will focus on the transcription of the gene into a usable RNA form. The goal of this lab is to evaluate the gene expression of β-1,3-glucanase (β- 1,3-glu) and cor15a in a transgenic plant of the species Arabidopsis thaliana when exposed to severe conditions. The plant responds to severe conditions by producing a large amount of salicylic acid which then triggers the plants cells to begin making β-1,3-glucanase. The β-1,3-glucanase promoter gene produces a hydrolytic protein which breaks down cell walls. The activity of this gene expression is evaluated using a combination spectrophotometry and fluorescence activity in the extracts which acts as an indicator of transcriptional activation. Materials and Methods Part I: Making Extract: To isolate the DNA and genes to be evaluated, they first need to be extracted from the plant material. First, 2 or 3 leaves from the plant sample were placed into a microcentrifuge tube. The extraction buffer (2-mercaptoethanol) was then added to the sample. Using a pestle, the plant leaves were ground in the microcentrifuge tube for 3-4 minutes until the solution turned a bright green. The tube was then centrifuged for 10 minutes to form a pellet of the plant material waste. The supernatant was then removed and evaluated in the latter parts. Part II: Protein Determination: This step is designed to determine the protein concentration in 20 uL of the newly formed plant leaf extract. First, the spectrophotometer was set to 595 nm. Then 5 mL of Coomassie blue reagent, the dye, was added to two test tubes. For the blank 20 uL of extraction buffer was added and to the second tube 20 uL of the extract was added. Both were vortexed and allowed to sit at room temperature for 5 minutes. The spectrophotometer was then zeroed using the blank tube and the concentration of the protein in the extract was then determined using a premade standard curve. From this data, the number of uL which contained 20 ug of the protein was then calculated for use in Part III. Part III: Fluorescence Determination:
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This note was uploaded on 02/16/2012 for the course BIOL_SCI 210-2 taught by Professor Somebody during the Winter '09 term at Northwestern.

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Lab 4 Transgenic Plants Gene Expression (1) - Transgenic...

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