BME103:T930 Group 10

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: Nolan Bidese Role: Research and Development Specialist Name: Evan Austin Role: Open PCR Machine Engineer Name: Aldin Malkoc Role: Open PCR Machine Engineer Name: Mikayle Holm Role: Experimental Protocol Planner Name: Coleen Fox Role: Experimental Protocol Planner LAB 1 WRITE-UP (Please finish by 11/7/2012) Initial Machine Testing The Original Design (Add image of the full OpenPCR machine here, from the Week 3 exercise. Write a paragraph description for visitors who have no idea what this is) Experimenting With the Connections When we unplugged (part 3) from (part 6), the machine ... (did what? fill in your answer) When we unplugged the white wire that connects (part 6) to (part 2), the machine ... (did what? fill in your answer) Test Run (Write the date you first tested Open PCR and your experience(s) with the machine) Protocols Polymerase Chain Reaction Polymerase Chain Reaction (PCR) is a technique used to amplify fragments of DNA. This allows researchers to see the base sequence of the DNA. It works by the DNA polymerase enzyme synthesizes a complementary strand of the fragmented DNA when mixed with primers that signal where the DNA sequencing should begin. When the DNA polymerase enzyme, MgCL2, dNTP’s, forward primer, and reverse primers are all added to the test tubes and placed in the PCR machine, the mixture is first heated to separate the double helix, then cooled to allow the primers to bind. After the primers bind, the polymerase completes the new complementary strands. The PCR machine then repeats heating and cooling cycles to multiply the fragmented DNA. After a couple hours, the now amplified segments of DNA can be analyzed to test for a cancer marker. Procedure: 1. Add a fragment of double stranded DNA, the pre-mixed Taq DNA polymerase, MgCl2, dNTPs, forward and reverse primers, and the DNA polymerase enzyme to test tube. 2. Place in PCR machine. 3. Set the following PCR cycle stages: Stage 1: 1 cycle, 95 degrees Celsius for 180 seconds (separate the DNA double helix) Stage 2: 30 cycles, 95 degrees Celsius for 30 seconds, 57 degrees Celsius for 30 seconds, 72 degrees Celsius for 30 seconds (separate the DNA double helix, primers bind to single strands of DNA at 57 degrees Celsius, DNA polymerase enzyme adds bases to singles strands of DNA segments at 72 degrees Celsius, cycle is repeated so the DNA is multiplied) Stage 3: 72 degrees for 180 seconds (final nucleotides are added) Final Hold: 4 degrees Celsius 4. Run the PCR machine. 5. After about 2 hours, the cycles should be completed. DNA can now be used for research and testing for a cancer marker. The PCR (GoTaq) Master Mix is advertised as a “ready-to-use solution” and it contains the Taq DNA polymerase, dNTPs, MgCl2 and reaction buffers. These substances are mixed at proper concentrations so the user can achieve a useable amplification of DNA segments by PCR. Reagent Volume Template DNA (20 ng) 0.1μL 10μM forward primer 0.5μL 10μM reverse primer 0.5μL GoTaq master mix 25.0μL dH2O 23.9μL Total Volume 50.0μL Samples Test tube 1 Positive Control Cancer DNA template Test tube 2 Patient 1 Replicate 1 ID: 30576 Gender: Male Age: 46 Test tube 3 Patient 1 Replicate 2 ID: 30576 Gender: Male Age: 46 Test tube 4 Patient 1 Replicate 3 ID: 30576 Gender: Male Age: 46 Test tube 5 Negative Control DNA Template Test tube 6 Patient 2 Replicate 1 ID: 96210 Gender: Male Age: 59 Test tube 7 Patient 2 Replicate 2 ID: 96210 Gender: Male Age: 59 Test tube 8 Patient 2 Replicate 3 ID: 96210 Gender: Male Age: 59 Flourimeter Measurements (Add your work from Week 3, Part 2 here) Research and Development Specific Cancer Marker Detection - The Underlying Technology (Add a write-up of the information discussed in Week 3's class) (BONUS points: Use a program like Powerpoint, Word, Illustrator, Microsoft Paint, etc. to illustrate how primers bind to the cancer DNA template, and how Taq polymerases amplify the DNA. Screen-captures from the OpenPCR tutorial might be useful. Be sure to credit the source if you borrow images.) Results (Your group will add the results of your Fluorimeter measurements from Week 4 here)