TPA Genotyping Weeks 6-11

TPA Genotyping Weeks 6-11 - TPA Genotyping I, II, III, IV...

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TPA Genotyping I, II, III, IV (Weeks 6, 8, 9, 11) Names _______________________________________________________________________ Lab Group Number ________________ Section ____________ Date__________________ Lab TA ____________________________________ TPA Genotyping Schedule TPA Genotyping I: Week 6 TPA Genotyping Prelab Step 1: Isolation DNA from cheek cells (individually) TPA Genotyping II: Week 8 TPA Genotyping Prelab Step 2: Initiate PCR using TPA-specific primers (individually) Step 3: Pour gel (in groups of four students) TPA Genotyping III: Week 9 Step 4A: Practice loading gel (individually) Step 4B: Run PCR products on agarose gel (in group of four students) TPA Genotyping IV: Week 11 TPA Genotyping Prelab Step 5: Interpretation and analysis (in groups of four students) Step 6: Questions (in groups of four students) Step 7: Submit for grading of Steps 5 and 6 (in group of four students)(4%) Purpose : In TPA Genotyping I, II, III and IV, students will isolate their own DNA from buccal washes using Chelex resin beads. Students will perform PCR on their DNA using primers for TPA (PLAT = tPA: tissue plasminogen activator) gene, a dimorphic gene, having two distinct alleles. The sizes of the PCR products from each of the two different alleles are 100 bp and 400 bp. Each students’ “alleles” will be revealed using agarose gel electrophoresis. Lab sections will compile the genotypes of all students and calculate genotypic and allele frequencies for TPA within the class. These data may be used in Bio 124. Stay tuned. Goals: To isolate student DNA from buccal cells
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To perform PCR on student DNA and visualize PCR products using agarose gel electrophoresis To determine students’ genotypes for TPA gene To reinforce basic genetic concepts To begin to consider class allele and genotype frequencies TPA Genotyping I (Week 6): Step 1 Step 1: DNA Isolation (individually) Materials: Sterile saline (0.9% NaCl) solution (12 ml/student in 15 ml conical tube) Chelex beads (resuspended) 1.5 ml sterile Eppendorf centrifuge tubes Markers Alcohol pads Needle Boiling water bath (boiling when needed) Tweezers Ice in ice bucket Instructions: Step 1: DNA Isolation from cheek (buccal) cells 1. Your mouth needs to be clean of food particles. If your mouth is not clean of food particles, please go to the restroom and rinse out your mouth with water. 2. Pour 13 ml of physiological saline from the conical tube into your mouth. Make sure you close the cap after you pour the saline into your mouth, so that the tube stays sterile and does not get contaminated. 3. Swirl the saline in your mouth for 2 minutes, as if you were using mouthwash. This will collect most of the loose cheek cells from the inner part of your mouth. 4.
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TPA Genotyping Weeks 6-11 - TPA Genotyping I, II, III, IV...

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