BME100 f2013:W1200 Group4 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

Madison CochranRole: Open PCR Machine Testing
Madison Cochran
Role: Open PCR Machine Testing
Joshua H. SarbolandiRole: Open PCR Machine Testing
Joshua H. Sarbolandi
Role: Open PCR Machine Testing
Pete Auerele-AleRole: Research and Development
Pete Auerele-Ale
Role: Research and Development
Hannah SwitzerRole: Research and Development
Hannah Switzer
Role: Research and Development
Lindsey MaciasRole: Protocol Planning
Lindsey Macias
Role: Protocol Planning

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design
The OpenPCR is a machine that preforms the porlymerase chain reaction(PCR). PCR is a method of copying DNA molecules by continuously raising and lowering the temperature of the DNA. OpenPCR starts by raising the temperature of a double strand of DNA to 95oC and holding it for 3 minutes, splitting the strand into two identical strands of DNA. The identical strands are then put though multiple cycles of denaturing, annealing, and extending that rapidly replicate the DNA. Once done with the cycles, the temperature is lowered and held at 4oC to end the reaction. As the OpenPCR is connected to a computer via usb, a software on the computer allows the user of OpenPCR to input the temperatures and holding times for denaturing, annealing, and extending the DNA. Once the software information is inputted, the OpenPCR begins the experiment and displays both the temperature and estimated time on the LCD screen as well as the software window.

Image:MIJMKI.jpg

The image above displays multiple viewing angles of OpenPCR. The original images of OpenPCR were obtained from multiple websites. Their citations are bellow.

Cheap PCR. Digital image. BioTechniques. N.p., n.d. Web. 23 Oct. 2013. <http://www.biotechniques.com/news/biotechniquesNews/biotechniques-301745.html>.

OpenPCR Ships worldwide. Digital image. MAKE. N.p., n.d. Web. 23 Oct. 2013. <http://makezine.com/2011/07/06/dna-is-now-diy-openpcr-ships-worldwide/>.

OpenPCR. Digital image. Ponko.com. N.p., n.d. Web. 23 Oct. 2013. <http://blog.ponoko.com/2011/07/09/open-pcr-—-an-open-source-diy-dna-kit-you-can-buy/>.


Experimenting With the Connections
Image:OpenPCR1.png

There were six different parts of the OpenPCR machine that we looked at when we disassembled it. This included the heating lid (part 1), the heating plate (part 2), the LCD screen (part 3), the power supply (part 4), the fan (part 5), and the circuit board (part 6). If all of the parts and connections are not working properly, then the machine as a whole will not work.

If the heating lid (part 1) did not work, the machine would not be able to maintain a specific heat, because it would be affected by the surrounding environment. If the heating plate (part 2) did not work, then the machine would not be able to control the temperature of the PCR tubes. If the power supply (part 4) did not work, then the machine as a whole would not be able to work.

When we unplugged the LCD screen (part 3) from the circuit board (part 6), the machine no longer displayed any data on the LCD screen.

When we unplugged the white wire that connects the circuit board (part 6) to the heating plate (part 2), the machine displayed a false reading of the temperature. It dropped from 27.8 degrees Celsius to -40.0 degrees Celsius.


Test Run

A test run was conducted at 12:55 PM on Wednesday October 23, 2013. The machine ran properly and at a good rate. By 1:55 PM, the OpenPCR had performed approximately 16 cycles. Ultimately, the machine passed the test run.




Protocols

Thermal Cycler Program
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  First Step: 95oC for 180 seconds                                 
  Second Step: 35 cycles of denaturing,annealing, and extending (in that respected order).
                      Denaturing: 95oC for 30 seconds
                      Annealing: 57oC for 30 seconds
                      Extending: 72oC for 30 seconds
 Last Step: 72oC for 180 seconds
 Hold: 4oC

DNA Sample: 50 μL each. Patient ID's from the UnderGrad Teaching Assistant(UGTA)

DNA Sample Set-up

Positive control: cancer DNA template Tube Label: A+ Patient 1 ID 51917 Replicate 1 Tube Label B1 Patient 1 ID 51917 Replicate 2 Tube Label C1 Patient 1 ID 51917 Replicate 3 Tube Label D1
Negative control: non-cancer DNA template Tube Label: A- Patient 2 ID 17539 Replicate 1 Tube Label B2 Patient 2 ID 17539 Replicate 2 Tube Label C2 Patient 2 ID 17539 Replicate 3 Tube Label D2


DNA Sample Set-up Procedure

  1. Step 1 Gather 8 tubes of 50 μL PCR reaction mix, 8 tubes of 50 μL diluted template + primers, and disposable transfer pipettes.
  2. Step 2 Label all reaction tubes according to table.
  3. Step 3 Add 50μL of each DNA sample Mix to the corresponding labeled reaction tube using a new pippete for each transfer to avoid cross-contamination between samples.


PCR Reaction Mix

  • The PCR reaction Mix contains Taq DNA polymerase, MgCl2, and dNTP's.


DNA/ primer mix

  • The DNA/ primer mix contains a sample of the the patient's DNA, forward primer, reverse primer.




Research and Development

PCR - The Underlying Technology

The Polymerase Chain Reaction (PCR) is a method of replicating strands of DNA. Using this relatively simple procedure, it is possible to copy, or "amplify", billions of copies of the DNA of interest so they can be used in a variety of applications, from detecting viral infections to manipulating DNA in genetic engineering or synthetic biology.

Components of a PCR Reaction

The process begins with the template DNA, the target DNA strand that is being copied. Primers are short, laboratory-made pieces of DNA that are designed to match to the segment of target DNA. One primer attached to the top strand at one end of the segment of interest, and the other primer attaches to the bottom strand at the other end. Another component of a PCR reaction is magnesium chloride. This introduces magnesium into the reaction, which activates the DNA polymerase and causes it to begin working. function of the taq DNA polymerase is to make copies of a cell's DNA before it divides in two. When the DNA polymerase encounters a primer that is paired with a longer piece of DNA, it attaches itself to the end of the primer and begins to add deoxyribonucleotides (dNTPs). Nucleotides are the building blocks DNA are made of. The DNA grabs nucleotides that are floating in the liquid around it and attaches them to the end of the primer, effectively doubling the pre-existing DNA strands.

The Steps of Thermal Cycling

Each step of the polymerase chain reaction is activated by a certain temperature. A PCR machine performs thermal cycling to raise and lower the temperature at the required times for the reactions to occur. For the first three minutes of the process, the tubes containing the components are heated to 95 degrees Celsius. At this temperature, the DNA double helix spends thirty seconds separating, creating two single-stranded DNA molecules, in a step called denaturing. At 57 degrees Celsius, the primers anneal for thirty seconds, pairing up with the targeted DNA strands. The extending stage occurs at 72 degrees Celsius and lasts for thirty seconds. When the DNA polymerase locates a primer attached to a single DNA strand, it adds complementary nucleotides onto the strand. The tubes are maintained at 72 degrees Celsius for three additional minutes to ensure all strands have completed the process. During the final hold, the temperature is lowered to four degrees Celsius, limiting the activity of DNA polymerase and basically freezing it for later study.

Nucleotides

DNA is composed of four types of molecules called nucleotides: adenine (A), thymine (T), guanine (G), and cytosine (C). These pair up through hydrogen bonding forming the "rungs" of DNA's double-helix "ladder". Certain bases always pair up with each other. For example, adenine and thymine always pair, and guanine and cytosine always pair. Although the base pairings are limited, the order possibilities of the bases are endless, and this is what creates the DNA code that determines every aspect of life.


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