BME103:T130 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 Name: Alexandra Brunelle Research and Development Specialist Name: Charul Singh Experimental Protocol Planner Name: Alex Hoffmann Experimental Protocol Planner Name: AJ Ciferno PCR Machine Engineer Name: Haley Gjertsen Research and Development Specialist 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 design for the PCR lid includes a see through glass lid along with a door opening like an oven's to slide the samples into place. The new lid will be double layered in order to fit twice the amount of samples. Therefore, there will be a shelf inside to allow the sliding of samples on the top and on the bottom when the door is open. We redesigned to be glass in order to allow better viewing of the samples. The machine will involve the same tightening screw technique of the previous PCR design. Before the glass redesign, we were unable to visually see how tight the lid was on the samples. This brought up the possibility of us tightening the lid too tight, thereby squishing the samples. Moreover, this led to the DNA sample tubes melting. Now that we have the transparent glass surface, we are able to see how tight the lid is, preventing us from harming the samples. Key Features A. Now designed to be on the side, serves as the tightening knob as in the previous design B. Glass oven-like door on the side C. Double layered to fit twice the amount of samples (32 samples) Areas marked by "*" indicate sections that will now be made from glass and therefore, transparent allowing for observation. New area for samples opens like an oven where the samples can be slid in. It will hold 32 samples instead of its original, 16. The sample trays will be placed one on top of an other when slid in onto the shelf. The glass is for seeing how tightly closed the oven lid is screwed on. Instructions 1. Unscrew glass "oven-like door" 2. Slide 16 samples onto the top shelf and 16 samples into the bottom shelf 3. Close door 4. Screw the door tight but not until it touches the sides of the sample ependorf tube 5. Run PCR as original Benefits With the new design the PCR machine can now run more samples at a time. Therefore, the efficiency of the system is increased. Also, there will now be an absolute way of determining whether the door is too tight on the tubes. This allows us to be positive that the PCR machine will perform the best test on the DNA samples. Protocols Materials Supplied in Kit Amount PCR machine with new glass top 1: $400 to $600 Fluorometer: (LED light glass stand) 1: $200.00 Connection cord for Computer to PCR 1: $20.00 Smartphone Stand 1: $5.00 Box 1: $5.00 Disk for ImageJ program 1: $0.50 Shelf and New glass lid 1: $100.00 Supplied by User Amount Computer 1: $1000.00 Pipettes 16: $0.25each Glass Slides 2: $1.00each Tubes 32: $4.00 Smartphone 1: $200.00 Water varies DNA samples varies DNA Reagent Materials Amount Patient's Template DNA 0.2 μL GoTaq master mix 50.0 μL dH2O 47.8 μL 10 μM Reverse Primer 1.0 μL 10 μM Forward Primer 1.0 μL Total 100 μL PCR Protocol Set up PCR Proceedures: 1. Label each tube differently and record label. 2. Insert reactants into their respective PCR tubes. 3. Connect assembled PCR machine to a computer. 4. Customize settings in program to two stages: Stage 1: 1 cycle, heat to 95 degrees Celsius for 3 minutes Stage 2: 35 cycles, heat to 95 degrees Celsius for 30 seconds; cool to 57 degrees Celsius for 30 seconds Stage 3: heat to 72 degrees Celsius for 30 seconds. 5. Start up the OpenPCR program previously installed on the computer. 6. Slide out the tray that you want to place your tubes in. 7. Place tubes in the slots. and close lid tightly. 7. Look through the glass on the side to lower the top of the lid to make sure it touches the top of the tubes. 8. Press "Start" to run the PCR machine. 9. Record data. Setting up the smart phone camera Set the smart phone camera menu to the following: 1. Inactivate the flash. 2. Set ISO to 800. 3. Set white balance to auto. 4. Set exposure to highest setting. 5. Set saturation to the highest setting. 6. Set contrast to lowest setting. DNA Measurement Protocol 1. Label each transfer pipette and Eppendrof tube to match the eight samples. 2. Pipette and Eppendorf tube for SYBR Green should be labeled with blue; DNA Calf Thymus Pipette and Eppendorf tube should be labeled with red; Label waste pipette. 3. Transfer each sample one at a time into the Eppendorf tube containing 400 mL of buffer with a matching label, using its respective pipette. 4. Take caution to not let pipettes touch each other. 5. Place two drops of SYBR Green (using the pipette with blue dot) onto the first two centered spots on the glass slide. 6. Place two drops of first diluted sample on top of the two drops of SYBR Green. 7. Turn on light and adjust glass slide so light goes directly through the drop. 8. With all equipment in the fluorimeter, Smartphone Operator will take pictures focused on the drop and give to image processor. 9. Using previously labeled waste pipette, remove drop from glass slide and discard waste. 10. Repeat steps 5 through 9 for each sample, using next two consecutive, centered spots on glass slide for next sample. Image Processing 1. Upload photo to ImageJ. 2. Click on ANALYZE, choose SET MEASUREMENTS, then AREA INTEGRATED DENSITY and MEAN GREY VALUE. 3. Click IMAGE, choose COLOR then SPLIT CHANNELS to create three files. 4. Choose image name that has "green" next to it. 5. Active Oval Selection by clicking on Menu bar. 6. Click and stretch oval around green or clear drop in image. Click ANALYZE and choose MEASURE. 7. Record sample number and measurements. 8. Draw another oval of the same size in the "green" file for the background above the drop to get the “noise”. Again, click ANALYZE and choose MEASURE. 9. Record sample name and measurements and use as background label. 10. Save measurements to Excel file. Research and Development Background on Disease Markers Transient Myeloproliferative Disorder of Down Syndrome is a combination of the diseases leukemia, down syndrome and acute myeloid. It is extremely rare and takes the life of a patient in the very early months of life. It is associated with the SNP (single nucleotide polymorphism) 121912500 on chromosome 21. http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=121912500 Our PCR machine can test for multiple DNA mutations such as cancer, like in previous experiments, and now Transient Myeloproliferative Disorder of Down Syndrome. Primer Design Transient Myeloproliferative Diorder of Down Syndrome: The forward primer of this disease is ACCGGCTGGTGGGCCCGCTG. The reverse primer that would correctly match the forward primer and not code for a diseased patient would be TGGCCGACCACCCGGGCGAC. The diseased allele and diseased patient will have a reverse primer TGGCCGACAACCCGGGCGAC. Therefore, a patient that contains the disease has an allele mutation within the pairing of G to C, it is instead G to A. This will give a positive on the PCR machine because it becomes single stranded due to the fact it cannot replicate properly. This, when we apply the SYBR green to it, will attach to only the single primers and the diseased patients will glow green. The replicated, normal strands, will appear clear when the SYBR green is added because it will not be able to attach to the replicated strands. Illustration Below is a visual representation of what happens during a Polymerase Chain Reaction: 1. The DNA is heated up to 95 degrees Celsius, splitting the strand apart. 2.The solution is cooled down to 50 degrees Celsius and primers are able to attach to the separated strands. 3.The solution is heated to 72 degrees Celsius and taq Polymerase attaches. 4. The DNA strands are replicated at 72 degrees Celsius. 5. The process repeats as the PCR machine heats up to 95 degrees. (Photo Credit: The images used here were borrowed from OpenPCR.) Bonus: Bayesian statistics allows you to analyze and use all available data and can help you under the limitations of diagnostic tests. In our case, it can be used to test a sample of patients for this disease. From the tests we can see the false positive rate, sensitivity the test has to the disease, and prevalence to better inform the public on the statistics of the disease within the population and the subject to failure the test has so the doctors can run more if necessary. D=disease positive T=positive test p=probability p(DIT)=[p(TID)*p(D)]/[(p(TID)*p(D))+(p(TI~D)*p(~D))]