BME100 f2013:W1200 Group13 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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Contents

OUR TEAM

Name: Samantha Brenna Role: initial machine testing
Name: Samantha Brenna
Role: initial machine testing
Name: Joseph Salerno Role: Protocols
Name: Joseph Salerno
Role: Protocols
Name: Briana Ybarra  Role: Protocols
Name: Briana Ybarra
Role: Protocols
Name: Brenda Vargas  Role: Research and Development
Name: Brenda Vargas
Role: Research and Development
Name: Scott Ashmore  Role: Research and Development
Name: Scott Ashmore
Role: Research and Development

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design
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OpenPCR with labels
OpenPCR with labels

The picture above shows what the OpenPCR machine looks like while running and connected to the computer. The machine case is made of wood panels which contains the actual hardware of the device. The smaller box located on the top is the lid of the OpenPCR machine. This contains a 16-tube PCR block which is where the test vials are placed in order to complete the test run. The top part of the lid has a heat plate that heats the vials and also seals the experiment. Inside of the main body of the OpenPCR machine there is a heatsink, processor, led screen, and copper wire connections. The processor has a circuit board that recieves the data from and computes the calculations. The heatsink dispersses the heat and the fan attached cools down the device in order to avoid overheating. The led screen displays the data specifically the numbers of tests run and temperature. The machine also has an USB port and power connection in order to power the device and connect it to the computer in order to run the tests.

Experimenting With the Connections

When we unplugged the LED screen (part 3) from the circuit board(part 6), the machine didn't display anything on the screen because this connection is what allows the power to connect to the led screen. When we unplugged the white wire that connects (part 6) to (part 2), the machine lost the ability to record data and to sense the heat change in the machine.


Test Run

Our OpenPCR machine passed the testing that we conducted in class. We began the testing at 12:50 pm, and at 1:40pm the machine had run 19 out of 35 tests. When we concluded the experiment the temperature according to the screen on the device was 95 degrees Celsius, and had an ETA of 48. The test ran very smoothly for the first time use and we didn't encounter problems with the machine not working properly. The device was plugged into the computer and then we opened the program in order to start the tests. Then after entering in the information given we began running the test. There were no issues with the program or the machine. )




Protocols

Thermal Cycler Program
Stage 1: 95°C for 3 minutes-Double helix unwinds into one long strand
Stage 2: (35 cycles of each)
1. 95°C for 30 seconds-Double helix breaks apart into two strands
2. 52°C for 30 seconds-Primers attach to target sequence
3. 72°C for 30 seconds-DNA polymerase begins constructing new strand
Stage 3: 4°C for 3 minutes-Keeps solution of pure taget sequence at stable temperature

DNA Sample Set-up

PC 1R1 1R2 1R3
NC 2R1 2R2 2R3

DNA Sample Set-up Procedure

  1. Step 1 Place each sample in its respective pipette tips. That is the positive control, negative control, patient 1, and patient 2.
  2. Step 2 Place the 4 pipettes inside the OpenPCR machine.
  3. Step 3 Run the machine for 1 cycle and record results.
  4. Step 4 Repeat step 3 untill the desired number of replications is reached.


PCR Reaction Mix

  • What is in the PCR reaction mix?

The PCR reaction , 50uL each. the mix contains Taq DNA polymerase, MGCl2, and dNTP's.

DNA/ primer mix

  • What is in the DNA/ primer mix?

Each mix contains a differant template DNA all tubes have the same forward primer and reverse primer.




Research and Development

PCR - The Underlying Technology
A Polymerize Chain Reaction is a way of making various copies of DNA molecules. A PCR reaction has 5 different components and each of these components have a different function. The first component is the template DNA, this is the sample that contains the sequence that we want to replicate. Then there's the primers,short pieces of synthesized DNA, that bond to the target sequence and are the starting point for replication. The Taq Polymerase physically constructs the new strand of DNA. In the reaction, Magnesium Chloride facilitates the bonding of the nucleotides and the Deoxyribonucleotides are converted to dNMP's by the magnesium chloride before bonding to another nucleotide.

At the beginning of the thermal cycling, the samples are heated for 3 minutes at a temperature of 95 degree Celsius, and the heat makes the double-helix unwind and separate into two single stranded DNA molecules. Afterwards, the temperature is brought down to 57 degrees Celsius, the temperature change makes the primer attach to the target sequence. Once again, the temperature is brought up, this time to 72 degrees Celsius, this activated the TAQ polymerase and attaches to the primer that is attached to the single DNA strand and adds complimentary nucleotides to the strand until it detaches. When adding complimentary base pairs, the adenine (A) bonds to thymine (T) and the cytosine (C) bonds to guanine (G). Now, we have a copy of the specific sample we wanted, in this case the cancer mutations. Doing this helps because the resulting product is almost entirely made up of the cancer mutation with very few impurities, and scientists can work and figure out what is wrong with that piece of DNA and try to solve the problem.


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