BME100 s2014:T Group14 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
The Original Design
A PCR machine is basically a photocopier for DNA. It splits apart the double helix by heating in order to make copies of it. For a polymerase chain reaction to take place a particular DNA sequence should be identified and then amplified by creating up to millions of copies of the specific strands. A PCR machine can go through many cycles of this process and can turn a strand of DNA into thousands of copies of the exact same single strands of DNA. The OpenPCR uses a display on the actual device and a USB port to connect to the computer in order to change the parameters of the PCR reaction we want to take place. The OpenPCR machine is basically a "Do-It-Yourself" kit for anyone to buy, make, and then use. Since it's you have to put together, this equipment is open to some errors if not put together right. This is why we tested the PCR machines before conducting our experiment.
Experimenting With the Connections
When we unplugged (part 3) from (part 6), the machine's display turned off; this is the power cable for the display.
When we unplugged the white wire that connects (part 6) to (part 2), the machine's display showed an incorrect temperature. We assume that this is the wire that connects the display and internal computer to the integral part of the device that measures the temperature of the PCR machine.
We preformed our test run of our OpenPCR machine on Thursday March 20, 2014. Set up was simple and went smoothly, we placed the empty test tubes into the machine and ran the machine. After setting up everything correctly, our machine ended up failing the test. The computer program projected that our the time for our reaction would extend way beyond the two hour time period and didn't even finish 1 cycle. This resulted in our machine's failure and was noted to not be used during the experiment.
Thermal Cycler Program
DNA Sample Set-up
This is the test tube set up we inserted into the OpenPCR system. We had two different subjects of DNA, one female and one male, and we labeled everything as follows on our 8 50 microliter test tubes.
dDNA -- the disease DNA
ndDNA -- the non-disease DNA
female subject -- 1a, 1b, 1c
male subject -- 2a, 2b, 2c
DNA Sample Set-up Procedure
1. Collect materials needed, which include: the PCR reaction mix in 8 test tubes with 50μL in each, 8 empty attached PCR tubes, 8 tubes of the Template DNA and the primer mix combined with 50μL in each tube, a box of disposable pipette tips, and a 200μL micropipettor. 2. Then cut the tubes in sets of 4. You need to do this in order for the tubes to fit in the OpenPCR machine. 3. Then the tubes will be labeled with the predetermined labels we named above with a permanent marker. 4. The tubes are placed in a test tube rack after being labeled. 5. Attach a tip to the micropipettor, and transfer 50μL of the PCR reaction mix into the positive control. Then, dispose the tip into the proper waste disposal. 6. Transfer the positive disease containing DNA and primer mix into the same tube and use a new tip and dispose it properly. 7. Repeat steps 5 and 6 for the negative control, patient 1, and patient 2, remembering to dispose the tip after each substance in each PCR tube. In total 100μL will be each test tube. 8. Place the tubes into the PCR machine, set up the parameters needed for the test run. 9. Get help from the TA in order to start the machine.
PCR Reaction Mix
DNA/ primer mix
Research and Development
PCR - The Underlying Technology
Heat Sync and Fan: The Heat Sync is the source of heat for the PCR testing. This piece is directly connected to the loading tray for the DNA samples. The fan is there to keep the insides of the PCR machine from overheating and failing.
Power Supply: The power supply directs power to all components of the PCR machine.
Motherboard/Brain Board: This piece holds all of the working of the PCR machine. The programming and functions of the PCR machine are carried out by the circuitry and the display of the LCD screen relies on this.
Polymerase Chain Reaction:
DNA Template: Strand of DNA that holds the target sequence for PCR testing.
DNA Polymerase: An enzyme that synthesizes strands of DNA. Taq DNA polymerase is commonly used. This enzyme is used for its heat resistance and can withstand the energy required for PCR targeting.
Primers: Primers are single strands of protein that attach the the split target DNA sequence and make way for the copying of the DNA sequence.
Nucleotides: Basic blocks of DNA (A,T,G,C) used for copying the target sequence of DNA.
Base pairs are nucleobases that are building blocks of DNA double helix and even contribute to the folded structure of the DNA because of the positioning of the hydrogen bonds between the bases. There are four base pairs in total: Guanine, Cytosine, Adenine, and Thymine. All of these base pairs only have one opposite that they can bond to in order to create the complementary double stranded DNA. The pairs are Adenine (A) to Thymine (T) and Guanine (G) to Cytosine (C). These pairs are not interchangable and is what makes copying DNA and the polymerase chain reaction possible.