BME103:T930 Group 3

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 Rohan Kumar Experimental Protocol Planner Kyle Stoneking Experimental Protocol Planner Lekha Anantuni R&D Scientist Joshua Eger Machine Engineer Austin Cuaderno Machine Engineer LAB 1 WRITE-UP Initial Machine Testing The Original Design The OpenPCR is a machine that is used to replicate DNA in order to amplify a specific gene. This machine primarily uses changes in temperature and various enzymes to facilitate the replication process multiple times. If the heating lid did not work, then DNA replication would not be possible. The heating lid covers the thermal cycler and holds the DNA down tight and fluctuates in temperature. The thermal cycler is where the samples are place and varies at set temperatures and times. The temperature change is crucial to the replication of DNA because only at certain temperatures dose the DNA properly replicate. The LCD display outputs the temperature of the thermal cycler. The power supply connects to an external power source. Experimenting With the Connections When we unplugged the lcd display wire(part 3) from the Arudnio chip(part 6), the screen turned off. Everything on the PCR was working fine expect there was no output on the display. When we unplugged the white wire that connects Arudino chip(part 6) to thermal cycler (part 2), the reading from the screen dropped to -40 degrees Celsius. We disconnected the wire multiple times and each time the screen displayed -40 degrees Celsius. Test Run We ran a test run on 10/25/2012. For this test we placed some empty PCR tubes into the machine and ran a simple test program on the Open PCR software. After the simple test was over we noticed that the display screen on the Open PCR lid matched very closely with what was displayed on our computer screen. The agreement between our computer screen and our PCR display meant that our diagnostic test was a success. Protocols Polymerase Chain Reaction We have been given 3 sets of samples of replicate DNA from two patients, to test for cancer makers. We labeled each sample carefully as to not cross contaminate the samples. We also used one positive control sample and one negative control, which contained no DNA,to give us a total of 8 samples. We mixed the samples together with Taq DNA polymerase, MgCl2, dNTP'S, forward primer and reverse primer. We used the PCR machine to replicate the DNA. After the PCR had finished replication, drops of the samples, mixed with syber green, were then placed in a fluorimeter. We used a Samsung Galaxy Nexus smartphone to take pictures of each drop. We then used image j to analyze the drops. Polymerase Chain Reaction Procedure: 1.)We received 3 replicate DNA samples each from two patients and One positive control and negative control sample for a total of 8 samples. The samples we were given were already in their PCR reaction mix form. This mix contained Taq DNA polymerase, MgCl2, dNTP's, forward primer and reverse primer. Each sample was 50 micro liters. 2.)We labeled 8 empty PCR tubes. For the first sample we labeled the 3 DNA samples 1A, 1B and 1C. For the second sample we labeled the tubes 2A, 2B and 2C. For the positive and negative controls, we labeled the tubes + and - respectively. 3.)Using one pipette per sample, to avoid contamination, we transferred the PCR reaction mix we were given to the PCR tubes. 4.)We then placed the samples in the PCR machine 5.)We set our PCR program to three stages. Stage one: 1 cycle, 95 degree Celsius for 3 minutes. Stage 2: 35 cycles, 95 degrees Celsius for 30 seconds, 57 degrees Celsius for 30 seconds, 72 degrees Celsius. Stage three: 72 degrees Celsius for 3 minutes and then hold at 4 degree Celsius. Sample one ID 43891: 48 Male Sample two ID 36890: 56 Female GO Taq DNA Mix Reagent Volume Template DNA (20 ng) 0.2 μL 10 μM forward primer 1.0 μL 10 μM reverse primer 1.0 μL GoTaq master mix 50.0 μL dH2O 47.8 μL Total Volume 100.0 μL Fluorimeter assembly Procedure: 1.) Flourimeter Measurements Area Mean Raw IntDen Sample - Noise Water Sample 30886 163.872 5061356 3890719 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 Sample Integrated Density DNA μg/mL Conclusion PCR: Negative Control 4528827 1.08 Positive PCR: Positive Control 8351858 2 Positive PCR: Patient 1 ID 43891, rep 1 5283304 1.26 Positive PCR: Patient 1 ID 43891, rep 2 4376083 1.05 Positive(borderline) PCR: Patient 1 ID 43891, rep 3 8144751 1.95 Positive PCR: Patient 2 ID 36890, rep 1 4190775 1 Negative(borderline) PCR: Patient 2 ID 36890, rep 2 5721560 1.37 Positive PCR: Patient 2 ID 36890, rep 3 5611447 1.34 Positive KEY Sample = Integrated Density = DNA μg/mL = Conclusion =