BME103 s2013:T900 Group7

BME 103 Spring 2013	Home People Lab Write-Up 1 Lab Write-Up 2 Lab Write-Up 3 Course Logistics For Instructors Photos Wiki Editing Help
Name: Amanda Seaney Role(s) Name: Carissa Henriksen Role(s)Protocol Name: Nathan Scheuer Role: Open PCR machine Engineer Name: Samantha Barker Role:Research and Development Scientist Name: student Role(s) Name: student Role(s) LAB 1 WRITE-UP Initial Machine Testing [[|thumb|right|1600x1250x|]] The Original Design The picture to the right shows the design of the open PCR machine used in class. This PCR machine is compact, inexpensive, and easy to use in comparison to professional grade PCR equipment making it ideal for student and school use. The machine is composed of an LCD screen, heating lid, mother board (circuit board),thermal conductor and a fan. Experimenting With the Connections Part 1 of the open PCR machine is the heating lid with a heating plate. This part heats the top part of the test tube as well as prevents heat from escaping. If this part doesn't work heat is likely to escape and the test tubes will not likely heat to the optimal temperature Part 2 is the test tube rack. This part hold the test tubes in place and acts as the main heating element for the test tubes. If this part does not work the test tubes will not heat to the optimal level Part 3 is a cable between the circuit board and the LCD display. If this connection fails the LCD will not function. Part 4 is a fan within the system. This part sucks the hot air out from the inside of the machine and prevents the system from overheating. Part 6 is a white wire between the circuit board and the rack. This wire is connected to a temperature sensor. Unplugging this wire will disable the temperature reading on the LCD display. Test Run On March 4, 2013 our PCR machine was first tested. We used machine "10" and downloaded the open PCR software on a mac laptop. The software did not download properly on my pc laptop. The system test ran normal without any issues. The time estimation on the software is not 100% accurate. Protocols Thermal Cycler Program For the reaction to take place, the machine needs to have a certain program set up to run. The program for the Thermal Cycler is as follows. This is also known as the initialization step: 1 Cycle at 95°C for 3 minutes 35 Cycles at 95°C for 30 seconds, 57°C for 30 seconds, 72°C for 30 seconds 72°C for 3 minutes Hold the mix at 4°C Each cycle consists of initialization, denaturation, annealing, and extension. The denaturation step is when the DNA is heated to a point at which the DNA template begins to melt, which leads to the double-stranded DNA coming apart into two single strands. Then the machine is cooled for the annealing step, which is when the primers attach to their respective sites on the single stranded DNA and begins the DNA formation. The fourth step, extension, takes place at a warmer temperature than annealing, but cooler than denaturation, and consists of the elongation of the DNA strands. DNA Sample Set-up Tube: A, Positive Control: Cancer DNA template Tube: B, Patient 1 ID: 43825, Replicate: 1 Tube: C, Patient 1 ID: 43825, Replicate: 2 Tube: D, Patient 1 ID: 43825, Replicate: 3 Tube: E, Negative Control: No DNA template Tube: F, Patient 2 ID: 12079, Replicate: 1 Tube: G, Patient 2 ID: 12079, Replicate: 2 Tube: H, Patient 2 ID: 12079, Replicate: 3 DNA Sample Set-up Procedure To keep the patient samples separate and to avoid contamination between samples, it is necessary for one to label the tubes by numbering them one through eight with a permanent marker. One should take up 25μL of patient DNA into the micro pipet. Following this, one should add it to the 25μL of the PCR reaction mix. After all 8 tubes are filled with 50μL one should place them into the PCR machine, which is connected to the computer. PCR Reaction Mix One must create a PCR reaction mix, in order to do this groups are presented with 8 tubes, which will be filled with 25μL of the following: Taq DNA polymerase dNTPs Magnesium chloride (MgCl2) Premium concentrations for the maximum amount of DNA produced by PCR DNA/ primer mix One must create DNA/ primer mix, in order to do this groups are presented with 8 tubes, which will be filled with 25μL of the following: Unique DNA template of each patient Forward and reverse primers (the same contents in each tube) Research and Development Specific Cancer Marker Detection - The Underlying Technology In order for a PCR machine to function in a way that is scientifically efficient, the machine must go through three different phases. It must go from 95ᵒC, to 57ᵒC, to 72ᵒC, and then repeat this cycle thirty times. At each stage, a microbiological change in the DNA occurrs. When the sample is heated to 95ᵒC, the hydrogen bonds that hold the two strands of DNA together are broken. It is known as unzipping DNA. This allows for the base pairs of the two DNA strands to be exposed. The sample is then dropped to 57ᵒC to allow for the primer to bind to the DNA template gene. The template DNA is the sample DNA that was extracted from the patient and the primer's function is to bind to the DNA and direct where the template DNA should be amplified. As the temperature increases to 72ᵒC, the Taq polymerase is activated. The polymerase is a protein that synthesizes the new DNA strand by using dNTP’s, the extra base pairs in the solution. The Taq polymerase grabs these extra base pairs and synthesizes the DNA strand that matches the template DNA strand. From here, the solution is reheated to 95ᵒC and all the steps repeat again. Each time this cycle is complete, the amount of replicated DNA strands will have an exponential growth in the amount of synthesized DNA. (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.)