BME103:T930 Group 2 l2

BME 103 Fall 2012	Home People Lab Write-Up 1 Lab Write-Up 2 Lab Write-Up 3 Course Logistics For Instructors Photos Wiki Editing Help
OUR TEAM Ryan Sullivan Research Development Scientist Miriam Y Acosta PCR Machine Engineer Ryan Keeney PCR Machine Engineer Juliana Ramos Experimental Protocol Planner Aaron Cornejo Experimental Protocol Planner LAB 2 WRITE-UP Thermal Cycler Engineering Our re-design is based upon the Open PCR system originally designed by Josh Perfetto and Tito Jankowski. System Design Our new and improved PCR Machine encompasses a redesign where we improved the inner materials of the machine. By doing so, we can decrease the time it takes to duplicate the dna samples, thanks to the new material, which helps the machine work faster and more efficiently when dealing with temperature changes. Key Features In addition, we made a change in the top lid of the Open PCR. We made one of the sides transparent to facilitate the tightening of the samples when they are placed in the rack. This way, one can easily see through the transparent side to check when they tightening is done. Instructions Protocols Materials Supplied in the Kit Amount Open PCR Machine 1 10 μM Forward Primer 16.0 μL 10 μM Reverse Primer 16.0 μL GoTaq Master Mix 800.0 μL dH2O 764.8 μL Eppendorf Tubes 16 Fluorimeter 1 Teflon Glass Slides 16 Allen Wrench 1 Operations Manual 1 Supplied by User Amount Screwdriver 1 Template DNA (20ng) 3.2 μL Micro-Pipetter 16 Camera Phone 1 PCR Protocol 1. Gather materials and assemble as shown in apparatus/manual 2. Label provided tubes with a number to indicate its sample. 4. With the micropipette, place the forward/reverse primer into the indicated tube. 5. Use a clean micropipette to place the master mix into the DNA/primer mixture in the tubes. 6. Transfer the 16 Eppendorf tubes containing DNA and Mix into the Open PCR Machine. 7. Open PCR program and input the number of cycles: 30. 8. Set the temperature and times into the program. - Denaturing temperature to 95°C. Time to 30 seconds. -Annealing temperature to 57°C. Time to 30 seconds. -Extending temperature to 72°C. Time to 30 seconds. 9. Close lid to commence process. 10. Start program. 11. When PCR is complete, ensue to the DNA Measurement Protocol shown below. DNA Measurement Protocol 1. Create 8 DNA template samples that will be the focus of the investigation and place them in the PCR Machine allowing them to complete the process and replicate. 2. Transfer each sample independently into its designated Eppendorf Tube with the 400mL buffer completely. Ensure that a single pipette is used per sample. 3. After setting up the fluorimeter, place 2 drops of SYBR Green onto the teflon slide followed by 2 drops of the sample you wish to use. (Again use the same pipette used to transfer the sample) 4. Align up the drop so the light is passing through it. 5. Take pictures of the drop using a camera or smartphone, these will later be uploaded to ImageJ for analysis. 6. Each slide is capable of handling 5 individual samples so simply place the drops in an empty space and repeat the process. 7. Also run drops from the scintillation vial as blanks using the same process. 8. Upload the pictures taken into Image J. 9. To analyze the images subtract the INTDEN measurement form the background from the INTDEN measurement form the drop and repeat for all trials. Research and Development Background on Disease Markers Sample A Sickle Cell Anemia rs35685286 [Homo sapiens] GGATGAAGTTGGTGGT--GAGGCCCTGG[A/G]CAGGTTGGTA--TCAAGGTTACAAGAC Chromosome 11- single nucleotide variation http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=35685286 Sample B Sickle Cell Anemia rs34430836 [Homo sapiens] AGGTGCTAGGTGCCTT--TAGTGATGGC[C/G]TGGCTCACCT--GGACAACCTCAAGGG Chromosome 11- single nucleotide variation http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=34430836 Disease Description Sickle cell anemia is an inherited blood disorder characterized primarily by chronic anemia and periodic episodes of pain. The underlying problem involves hemoglobin, a component of red blood cells. Hemoglobin molecules in each red blood cell carry oxygen from the lungs to body organs and tissues and bring carbon dioxide back to the lungs. In sickle cell anemia, the hemoglobin is defective. After hemoglobin molecules give up their oxygen, some may cluster together and form long, rod-like structures. These structures cause red blood cells to become stiff and assume a sickle shape. Unlike normal red cells, which are usually smooth and donut-shaped, sickled red cells cannot squeeze through small blood vessels. Instead, they stack up and cause blockages that deprive organs and tissues of oxygen-carrying blood. This process produces periodic episodes of pain and ultimately can damage tissues and vital organs and lead to other serious medical problems. Normal red blood cells live about 120 days in the bloodstream, but sickled red cells die after about 10 to 20 days. Because they cannot be replaced fast enough, the blood is chronically short of red blood cells, a condition called anemia. Inheritance Sickle cell anemia is an autosomal recessive genetic disorder caused by a defect in the HBB gene, which codes for hemoglobin. The presence of two defective genes (SS) is needed for sickle cell anemia. If each parent carries one sickle hemoglobin gene (S) and one normal gene (A), each child has a 25% chance of inheriting two defective genes and having sickle cell anemia; a 25% chance of inheriting two normal genes and not having the disease; and a 50% chance of being an unaffected carrier like the parents. Source: http://www.ornl.gov/sci/techresources/Human_Genome/posters/chromosome/sca.shtml Primer Design Sample A rs35685286 [Homo sapiens] Primer--CTCCGGGACCTGTCCAACCAT Reverse Primer-- GAGGCCCTGGACAGGTTGGTA Sample B rs34430836 [Homo sapiens] Primer--ATCACTACCGGACCGAGTGGA Reverse Primer--TAGTGATGGCCTGGCTCACCT Illustration Sample A This image illustrates the primer attaching to the reverse primer. The primer that is created attaches to the section of DNA that is mutated and is then able to be replicated with PCR, because it will allow the Taq polymerase to begin attaching nucleotides. Sample B This image illustrates the primer attaching to the reverse primer. The primer that is created attaches to the section of DNA that is mutated and is then able to be replicated with PCR, because it will allow the Taq polymerase to begin attaching nucleotides.