BME103:T130 Group 15 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: Alyssa Alexander Research & Development Name: Student Role(s) Sichun Ai protocols planner Name: Student Role(s) Name: Student Role(s) 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 Key Features Instructions Protocols Polymerase Chain Reaction Polymerase chain reaction is basically molecular photocopying and the process or technique used to make copies of small segments of DNA because it only targets specific segments of the DNA and that's what makes it useful. PCR works by mixing two DNA fragments, also known as primers which are about 20 bases long. The mixture is then heated and denatured and then the primers bind to their complementary sequences on the separated strands. Then, the polymerase extends primers into new complementary strands and it goes through about 30 cycles. PCR products are useful and can be used in many experiments like DNA fingerprinting and detection of viruses. (Jim Dorsey) Polymerase Chain Reaction. http://www.contexo.info/DNA_Basics/polymerase_chain_reaction.htm. Last accessed 11/01/12.) Materials Supplied in Kit PCR Machine 1 Instruction Manual 1 Extra screws/bolts/nuts 5 each USB Cable 1 Extra Battery 1 Power Cord 1 Supplied by User Computer 1 Smartphone with Camera 1 Pipettes 12 Eppendorf Tubes 8 Distilled Water As much as needed SYBR Green I 5 mL Smartphone holder 1 Fluorimeter 1 Image J Software 1 GoTaq Mastermix 1 Glass slides As many as needed (1) Calf Thymus 50 μL PCR Protocol Steps to Amplify DNA Samples Collect three replicate DNA samples from two patients. (was provided already) Create a new program on the Open PCR system (connected to the computer) Create three stages Stage 1: 1 cycle, 95°C for 3 minutes Stage 2: 30 cycles for 95°C for 30 seconds, 57°C for 30 seconds, 72°C for 30 seconds Stage 3. 72°C for 3 minutes Final hold: 4°C The DNA samples are 50 μL each, get the patient's ID and label the the each tube. PCR reaction mix - Mix contains Taq DNA polymerase, MgCl2, dNTP's, forward primer, and reverse primer. The primers are artificial DNA, designed to match the chain of DNA we want. Taq polymerase is the enzyme that binds to the end of the new chain and recreates the separated DNA. Mgcl2 binds to Taq as a co-factor and helps Taq to function appropriately, and affects the speed of the Taq binding to the loose strands. dNTp's is dioxnucleotidetriphosphate. this is what is used to recreate the second DNA strands. The 8 tubes of mixtures will then go through the cycles in the PCR system. During each step of the thermal cycling, the DNA is unzipped and heated to 95°C to break the H-bonds between the 2 strands. This exposes the part we want in this lab experiment. Then, the primer binds to the trage we want without cancer marker, this primer won't bind. Next, the temperature will be dropped to 57°C in order to bind the primer. Later, it is heat it back up to 72°C with the Taq to reform and duplicate DNA strands. Finally, this thermal cycling is repeated for amplification and add dye that binds specifically to DNA for detection. The Components of the GoTaq® Colorless Master Mix "dNTP's, MgCl2, and reaction buffers at optimal concentration for efficient amplification of DN templates by PCR." Volumes Used for Mixture Table 1 Reagent Volume Template DNA (20 ng) 10.2 μL 10 μM reverse primer 1.0 μL dH2O 47.8 μL 0 μM forward primer 1.0 μL GoTaq master mix 50.0 μL Total Volume 100.0 μL DNA Samples (8) Positive Control: Cancer DNA Template Tube label A Replicate 1 Tube Label 1-1 Patient 1 ID: 27762, F, Age: 52 Replicate 2 Tube Label 1-2 Patient 1 ID: 27762, F, Age: 52 Replicate 1 Tube Label 1-3 Patient 3 ID: 27762, F, Age: 52 Negative Control: No DNA Template Tube Label B Replicate 2 Tube Label 2-1 Patient 2 ID: 59484, F, Age: 45 Replicate 2 Tube Label 2-2 Patient 2 ID: 59484, F, Age: 45 Replicate 2 Tube Label 2-3 Patient 2 ID: 59484, F, Age: 45 DNA Measurement Protocol Research and Development Background on Disease Markers Primer Design Illustration