BME100 f2013:W1200 Group5 L4
|BME 100 Fall 2013|| Home |
Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
Course Logistics For Instructors
Wiki Editing Help
LAB 1 WRITE-UP
Initial Machine Testing
The Original Design
When we unplugged part 3 the screen from the mother board, the screen on the machine turned off.
When we unplugged the white wire that connects the mother board to the PCR block, the temperature reading on the machine lowered, which caused the reading to be incorrect and read that it was at -40°C.
On October 23, 2013 we tested the Open PCR machine and found that it functioned well. The temperatures fluctuated somewhat when it went through the heat cycles, but usually only by about .1°-.2°. There was somewhat of a lag between the readings on the machine and the readings on the computer as well. As for the timing of the cycles, the machine worked correctly and properly and cycled through the temperatures at the correct time. We started at 12:55pm and at 1:40 pm our machine had gone through 15 cycles.
The PCR machine begins by warming up to 100 degrees Celsius to begin the process. Then, the initial step takes place for three minutes at 95 degrees Celsius. This is done to heat up all the DNA strands, serving as a pre-denaturation step. Then the next three steps are repeated for a total of 35 cycles. The first step in this cycle is the denaturation cycle, and it is done at 95 degrees Celsius for 30 seconds. This is done to separate the double stranded DNA to single strands, forcing all reactions from enzymes to stop. The second step of the cycle is the annealing process, and it is done at 57 degrees Celsius for 30 seconds. This is when the polymerase attaches and begins to copy the DNA template. The last step of the cycle is the extending process, and it is done at 72 degrees Celsius for 30 seconds. This is when the polymerase couples to the primer on the 3' side, adding the bases that are complementary to the DNA template. The denaturation, annealing, and extension cycles are repeated for a total of 35 cycles. Then, the final step at 72 degrees Celsius for three minutes ensures that all the DNA goes through the extension process before the PCR machine cools down again. Finally, the PCR machine cools down to 4 degrees Celsius to ensure that all the single strands of DNA bond into double strands once again.
DNA Sample Set-up
Research and Development
PCR - The Underlying Technology
Components of a PCR Reaction
PCR: The Process of Thermal Cycling
The template strand containing the target sequence. (http://learn.genetics.utah.edu/content/labs/pcr/)
To initiate thermal cycling, the template DNA strand must be heated at a temperature of 95°C over 3 minutes. The heat will cause the double helix to straighten and then denature for 30 seconds. When the DNA strands denature, they break apart at the nucleotide bonds, leaving each strand with its own set.
Next, the temperature is reduced to 57°C for 30 seconds (Anneal Phase) to allow the primers to attach to the target sequence on each DNA strand.
Once the primers have attached, temperature is raised to 72°C for the Extend Phase and in 30 seconds DNA or Taq polymerase binds to the primers and becomes activated by the Magnesium Chloride. Holding the temperature at 72°C over the next three minutes allows the polymerase to synthesize the entire complementary DNA strand.
The solution can then be held at 4°C and the final product becomes two complete sections of the target sequence.
This completes the first cycle of PCR however, more cycles need to be run to isolate the target sequence and amplify it to testable levels. With each complete cycle the target sequence will multiply exponentially and will exist separate from the extra template DNA sequences that you do not wish to study.