BME100 s2014:T Group8 L4

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BME 100 Spring 2014 Home
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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: Nicole PlacheckiRole(s)
Name: Nicole Plachecki
Role(s)
Name: Colton TuckerRole(s)
Name: Colton Tucker
Role(s)
Name: Omar BenitezRole(s)
Name: Omar Benitez
Role(s)
Name: Hooyoung KimRole(s)
Name: Hooyoung Kim
Role(s)

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design

Internal Design of an Open PCR Machine.
Internal Design of an Open PCR Machine.

The openPCR is an over-glorified oven. It's purpose is to heat the dna to specific temperatures very precisely in order to achieve PCR. The openPCR oven has a display that it uses for user-interface, and a usb port which is used to connect to the computer and change parameters. Other than that, the openPCR machine is a simple heating pad connected to a heatsink/fan, and the heatsink and fan are connected to an arduino. What the arduino does is that it takes the temperature from the heating pad, and depending on what the desired temperature is, it either makes the heating pad hotter, or to cool the heating pad, it speeds up the fan on the heatsink. In order for the arduino to do this, it has constant temperature monitoring.

Image Source: http://openwetware.org/wiki/Image:300px-Labeled_OpenPCR.png


Experimenting With the Connections

When we unplugged the display screen (part 3) from the circuit board (Part 6), the machine's display turned off; this was a power cable for the display.

When we unplugged the white wire that connects the circuit board (Part 6) to the heating container (Part 2), the machine's display stopped showing a accurate temperature. This wire then, was the wire that told the arduino what the temperature was, and by association, told the display what the temperature was.


Test Run

The Open PCR machine was first tested by our group on March 20, 2014. As a trial run we let the Open PCR machine run for about eight cycles, which took approximately thirty minutes to complete. The specific machine that our group used complied with the partnered computer software and our custom heating/cooling protocol, and the trial run ran without errors or significant delays and thus proved the machine was completely functional and therefore passed. There were also no visible problems on the inside when we opened it up nor on the outside/heating pad.



Protocols

Thermal Cycler Program


DNA Sample Set-up

Patient 1 Patient 2 Controls
Sample 1 P1A P2A +C+ (Positive Control)
Sample 2 P1B P2B -C- (Negative Control)
Sample 3 P1C P2C

DNA Sample Set-up Procedure

1. Obtain 8 empty PCR tubes, tube-holding rack, positie control DNA, negative control DNA, patient DNA (3 from each patient) and a pipette with disposable tips.
2. Cut the empty tubes into two sets of 4 to fit the machine.
3. Label each tube with the pre-determined labels.
4. Place tube in the rack and attach a tip to the pipette.
5. Transfer 50μL of PCR reaction mix into the positive control-labeled tube. Dispose of the tip into proper waste bin.
6. Transfer the "+" DNA and primer mix into this tube using a new tip. again, dispose of this tip properly.
7. Repeat Steps 5 and 6 using the negative control, patient 1 DNA in all 3 replications, and Patient 2 DNA in all 3 replications. Ensure that the tip is changed for every new substance being put into any PCR tube. Each tube should end up with 100μL total.
8. Secure lids on each tube and place them into the PCR machine.
9. Get the help of TA to properly start the machine.


PCR Reaction Mix

  • A PCR Reaction MIx contains a buffer, water, deoxyribonucleotides (dNTPs), DNA primers, the Taq DNA Polymerase.

DNA/ primer mix

  • The DNA/ Primer mix contains a primer (RNA strand of about 20 nucleotides) and the subject's DNA (or the control mixture).





Research and Development

===PCR - The Underlying Technology===

Functions of PCR Components

Each PCR reaction starts with the template DNA. These function as the master copy for the gene to be replicated. The primers attach to opposite ends of the two strands once the DNA has been denatured, and serve as an attachment point for the Taq Polymerase. Taq Polymerase, an enzyme important to PCR because of the high temperature, travels along the template DNA--and later the new DNA copies--adding nucleotides to the primer to replicate the DNA. THe deoxyribonucleotides are DNA-building blocks that make up the Code that DNA is comprised of.


Nucleotides and Base Pairing

Deoxyribonucleotides in DNA and ribonuculeotides in RNA are the components of the biological code that makes up genes. There are four nucleotides for DNA: Adenine (A), Thymine(T), Cytosine (C), Guanine (G). When combined into dual-stranded DNA, Adenine bonds with Thymine and Cytosine bonds with Guanine.


Thermal Cycling

Thermal Cycling makes PCR possible. Once all components have been placed in the PCR tubes and the machine started, it heats up to 95°C for three minutes to get up to temperature and start to unravel the DNA double strand. The 95°C is maintained for another 30 second during the Denaturing Phase to separate the two strands on DNA. Then, the temperature drops to 57°C for 30 seconds in the Annealing Phase so that the primers can attach at the desired codon for the target gene of replication. The next 30 seconds are spent at 72°C for 30 seconds in the Extending Phase, where Taq Polymerase attaches to the end of the primer and begins copying DNA. In the Final step, maintaing 72°C for 3 more minutes, Taq Polymerase finishes relication. The Final Hold at 4°C allows all genes and components to settle and maintain their structure.






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