BME100 f2013:W1200 Group15 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: Patrick McFarlandProtocol Planning
Name: Patrick McFarland
Protocol Planning
Name: Zac RoyOpen PCR Machine Testing
Name: Zac Roy
Open PCR Machine Testing
Name: Taylor OlveyOpen PCR Machine Testing
Name: Taylor Olvey
Open PCR Machine Testing
Name: Gregory BerghorstResearch and Development
Name: Gregory Berghorst
Research and Development
Name: Tameem JamalOpen PCR Machine Testing
Name: Tameem Jamal
Open PCR Machine Testing

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design
Image:Open PCR Machine Group 15.png Source(https://www.google.com/search?q=Open+PCR+machine&source=lnms&tbm=isch&sa=X&ei=ZyxoUsWsO6bwiwLEooCYAw&ved=0CAcQ_AUoAQ&biw=1920&bih=961#facrc=_&imgdii=_&imgrc=Ct82Mj8rloOURM%3A%3BYGkEtXwApqO_fM%3Bhttp%253A%252F%252Fopenwetware.org%252Fimages%252F4%252F41%252FPCR_Machine.png%3Bhttp%253A%252F%252Fopenwetware.org%252Fwiki%252FBME103%253AT130_Group_7%3B1377%3B1027). This device is known as the OpenPCR is used for the amplification of DNA. The samples that are placed in the machine consist of DNA, two different primers, DNA polymerase, and nucleotides. Minimal DNA is needed for the reaction so hair and small blood samples would suffice. Once a proper PCR sample is ready, it is put inside to machine where it will be put through a cycle of heating and cooling. The sample will then be heated to 95 degrees Celsius, causing the DNA to "unzip". It is then cooled down to 50 degrees Celsius so that the primers can react with the DNA. Heated up once again to 72 degrees Celsius, the Dna polymerase and begins to add complimentary nucleotides to the stands. This cycle will repeat 30 times until there are over a billion copies of the desired DNA.


Experimenting With the Connections

When we unplugged the LCD Display from the Circuit Board, the machine will not be able to monitor the heat source that is being applied to the DNA samples.

When we unplugged the white wire that connects the Circuit Board to the Heating Lid, the machine lost all power and would not heat up the DNA samples to the correct temperature.


Test Run

The date we first tested Open PCR was October 23, 2013. The machine is fairly simple and easy to work with. The computer program for Open PCR was fast paced and very easy to understand. Little, if no, confusion using this machine occurred. The Open PCR machine creates a new experiment fairly quickly, and has absolutely no difficulty to operate the device.
Started at 12:32pm and by 1:32pm was at cycle 17.




Protocols

Thermal Cycler Program
In the first step, the sample is heated to 95 degrees C for three minutes. This allows the DNA to disassociate and separate. Next, Helicase unzips the DNA, and the primer is created, ready to be copied. The machine is then cooled to 57 degrees, which at this point, the DNA tries to go back together, however the primers far outnumber the DNA, which then attaches to the DNA recreating the template DNA. Finally, the solution is heated to 72 degrees, which the polymerase is activated, and extends the DNA sequence until the template is fulfilled.

DNA Sample Set-up

Positive Control: PCC Patient 1 Sample 1: P11 Patient 1 Sample 2: P12 Patient 1 Sample 3: P13
Negative Control: NCC Patient 2 Sample 1: P21 Patient 2 Sample 2: P22 Patient 2 Sample 3: P23


DNA Sample Set-up Procedure
1. Receive 8 sample tubes from Professor, containing 50μL of PCR reaction material
2. Label Samples according to table, to avoid swapping results
3. Put appropriate DNA sample into the accordingly labeled tube. Use a new pipette tip for each test tube to avoid cross contamination
4. Place the 8 sample tubes into the Thermocycler
5. Follow Thermocycler instructions above


PCR Reaction Mix
1. Taq DNA polymerase
2. MgCl2
3. dNTP


DNA/ primer mix
1. Sample of patient's DNA
2. Forward Primer
3. Reverse Primer





Research and Development

PCR - The Underlying Technology

Components of the PCR reaction and what they do.
PCR, and DNA for that matter would not be possible without dNTP’s which are the base pairs of a DNA molecule, including ATCG. With the building blocks of the DNA molecule identifies, the next important component of PCR is the Template DNA, which contains the target sequence of DNA to be copied. Once identified, primers are created through DNA replication, which are the match to the DNA template. At this point, the PCR can now commence, which through the help of taq polymerase and Magnesium chloride, the DNA reassembles via taq polymerase and regulated and stabilized through Magnesium chloride.

Steps of Thermal cycling
PCR is entirely based upon the regulation of temperature, and through many different cycles, the DNA is replicated. In the initial step, the sample is heated to 95 degrees C for three minutes. This allows the DNA to disassociate and separate. Next, Helicase unzips the DNA, and the primer is created, ready to be copied. The machine is then cooled to 57 degrees, which at this point, the DNA tries to go back together, however the primers far outnumber the DNA, which then attaches to the DNA recreating the template DNA. Finally, the solution is heated to 72 degrees, which the polymerase is activated, and extends the DNA sequence until the template is fulfilled.

Structure of DNA
DNA is structured in a double helix structure, composed of nitrogen bases called dNTP’s. These bases are what all DNA is composed of, ACTG. Adenine binds to Thymine, and Cytosine bonds to Guanine, with these set pairs, copies can easily be made by finding the complementary strand as seen in PCR.




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