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Unformatted text preview: Exercise 7 Analysis of PCR of TPA-25 and Bacterial Transformation 95 EXERCISE 7 DETECTION OF PCR PRODUCTS AND ANALYSIS OF THE ALU INSERTION POLYMORPHISM and RESULTS OF BACTERIAL TRANSFORMATION * GENOTYPING AND THE TPA-25 LOCUS * ANALYSIS OF FLUORESCENT REPORTER GFP IN TRANSFORMED BACTERIAL COLONIES Equipment and supplies needed Analysis of TPA-25 amplification agarose P-20 pipetman and tips electrophoresis chambers Blue tracking dye gel buffer (TBE) Ethidium bromide Erlenmeyer flask graduated cylinder balance, weighing pans and spoons microcentrifuge microwave oven padded gloves pGEM DNA standards power supplies image analysis system UV light computers Analysis of Bacterial Transformation bacterial plates UV illuminator Harvest of F2 Seeds filter paper circles forceps The purpose of this exercise is to analyze relative migration of the PCR fragments in an agarose gel, to analyze the results of the bacterial transformation and to harvest and germinate the F2 fast plnts. PART I: Analysis of PCR amplification Preparation of a 1.5% agarose gel (6 students will use 1 gel). 1. Prepare 40 ml of a 1.5% agarose solution. Calculate the amount of agarose required in the space below. 2. Weigh out the required amount of agarose and place in an Erlenmeyer flask. Exercise 7 Analysis of PCR of TPA-25 and Bacterial Transformation 96 3. Add 40 ml of electrophoresis buffer and proceed as in Exercise 4 to heat the solution in the microwave until dissolved. 4. Allow the agarose to cool slightly (5 minutes) before pouring into the chamber. Be sure to insert the comb. Preparation of the samples 1. While the gel is cooling, obtain your PCR tube from your TA and add 4l of DNA dye. 2. Centrifuge for 10 seconds to collect the dye in the bottom of the tube. 3. When your gel is completely cooled, remove the rubber ends and comb carefully and place the gel in the electrophoresis chamber containing about 300ml electrophoresis buffer. Be sure the gel is submerged in the buffer and that the wells are located at the end of the gel closest to the black electrode. 4. Remove 20l of the blue solution and load your sample into the gel....
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