BME100 s2014:W Group11 L4
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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
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LAB 1 WRITE-UP
Initial Machine Testing
This is an Open PCR machine with the access wall removed. This machine is the thermal cycler for the PCR process. Test tubes with a sample of DNA and PCR solution are placed in the tray at the top. The machine first heats up to a temperature below boiling point at which DNA denatures. It then cycles through temperatures so that primers in the PCR solution can bind with the ends of the target sequence of DNA. Polymerase can add corresponding nucleotides to form a complementary strand based on the primer at a lower temperature. The temperature is raised and the new strands denature. The process is repeated several times until the target sequence of DNA is sufficiently amplified. The metal box structure in the left side of the machine is the heating unit. The metal box on the right cools the samples.
When we unplugged the LCD screen (part 3) from the Open PCR Brains board (part 6), the machine stopped displaying any information on the screen. The screen stayed illuminated, but had no characters on it.
When we unplugged the white wire that connects the Open PCR Brains board (part 6) to the PCR Core (part 2), the machine changed the temperature reading of the PCR block on the LCD display from 24.4 degrees Celsius to -40 degrees Celsius.
We first tested Open PCR on March 19, 2014. The first machine we tested was found to have many disconnected wires. It gave highly unrealistic readings for room temperature (69.4 degrees Celsius). The next machine we tested was found to have one disconnected wire that we reconnected. It then appeared to function adequately. Its readings for internal temperature was 24.4 degrees Celsius, which matched the temperature of the lid. However, the test-run using the Open PCR software failed. The machine carried out the first step correctly. The lid heated to 100 degrees Celsius, and the machine heated the test tubes to 95 degrees C for two and a half minutes. After the initial step, the machine was unable to complete the first cycle. Its temperature decreased to 77 degrees C, then started slowly increasing again. After 20 minutes, it had gotten back up to 82.5 degrees C. It never got to the annealing stage or the extending stage. The test-run with the second Open PCR machine failed. By the end of the test, the machine had spent 40 minutes slowly increasing from 77 degrees to 83.8 degrees Celsius, never completing the first cycle.
Thermal Cycler Program
Step 2: Plug in the Open PCR machine to an outlet and plug it into the device with the software installed on it.
Step 3: Place empty PCR tubes in the machine and make sure the lid is closed and tightened.
Step 4: Create a new program on the Open PCR software and add a new experiment with heating lid at 100 degrees Celsius, initial step at 95 drees Celsius for 2 minutes. Number of cycles set to 35, denature at 95 degree Celsius for 30 seconds, anneal at 57 degrees Celsius for 30 seconds, and Extend at 72 degrees Celsius for 30 seconds. Final step should be 72 degrees Celsius for 2 minutes and final hold should be 4 degrees Celsius.
Step 5: Start the run with the software as you've programmed it.
Step 6: If machine successfully finish write Pass on machine. If not write fail.
Step 7: Come up with a label plan for the DNA samples that we will be testing once the machine has been passed.
The 50 μL sized mix contains a combination of a nonrecombinant modified form of Taq DNA polymerased, dNTPs, and MgCl2.
DNA/ primer mix
The 50 μL sized samples each contain a different template DNA. Each of the samples contain tubes which have the same forward primer and reverse primer.
Research and Development
PCR - The Underlying Technology
Components of a PCR Reaction
PCR, or Polymerase Chain Reaction, is a technique used to replicate DNA and/or amplify certain segments of DNA. This is extremely useful in a variety of situations such as detecting viruses and bacteria, genotyping, and monitoring an environment. Components necessary for carrying out PCR include template DNA, primers, Taq Polymerase, and Deoxyribonucleotides (dNTPs). Template DNA is the source of DNA that is to be replicated, either in full or a specific segment. Primers are short, designed segments of DNA that attach to the specific starting point of the target sequence of DNA. Since DNA polymerase (an enzyme that adds to DNA) can only attach to and add onto an existing strand of DNA, primers are required. Taq Polymerase is a commonly used type of DNA Polymerase that has a function of adding nucleotides to the end of the primer based on the template strand, thus creating a new strand that is complementary to the original. This entire process is dependent upon deoxyribonucleotides, which are the building blocks of DNA. There are four distinct dNTPs, adenine, thymine, cytosine, and guanine. These dNTPs form the information-conveying part of the DNA strand. Free-floating dNTPs are what the Taq polymerase uses to form complementary strands of DNA.
Steps of Thermal Cycling
PCR requires many steps during which the temperature of the reaction is changed. In the initial step, the temperature of the reaction is raised to 95 degrees Celsius and held there for 3 minutes. This step is necessary if the polymerase used in the reaction is heat-activated. Next is the denaturing step. The PCR mixture is kept at 95 degrees Celsius for 30 seconds. This temperature is hot enough that the DNA denatures. Its strands separate from each other, leaving two strands of unbonded, exposing the dNTPs. The temperature is ow enough that the polymerase doesn't denature. The next step is annealing, which takes place at 57 degrees Celsius over the course of 30 seconds. In this stage, free-floating primers attach to their complements on each DNA strand. Taq polymerase attaches to the site of the primer's attachment. During the extending step, the temperature is changed to 72 degrees Celsius for 30 seconds. During extension, the Taq polymerase adds free-floating dNTPs from the PCR solution onto the primer, creating a new strand of DNA that is complementary to the original strand of DNA. For the final step, the temperature is held at 72 degrees Celsius for 3 minutes. This step ensures that the new strands of DNA have finished elongating. Lastly, the temperature of the reaction is brought down and held at 4 degrees Celsius. This allows the reaction to be stored for a short period of time.
DNA Base Pairs
Genetic information is stored in DNA through the sequences of four different nucleotides, dNTPS. These dNTPs consist of adenine (A), thymine (T), cytosine (C), and guanine (G). Their base pairing is made using hydrogen bonds that connect the two strands of a DNA double helix together and only anneal in the forms of A-T and C-G.