BME100 f2013:W900 Group14 L4

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BME 100 Fall 2013 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: Rachael BrardRole(s)
Name: Rachael Brard
Role(s)
Name: Andrew CableRole(s)
Name: Andrew Cable
Role(s)
Name: Austin DavisRole(s)
Name: Austin Davis
Role(s)
Name: Omar EltohamyRole(s)
Name: Omar Eltohamy
Role(s)

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design
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reviews_pcr_f.jpg
PCR stands for polymerase chain reaction. The PCR machine depicted here is a biochemical technology that when used properly can amplify a selected piece of DNA to make millions of copies. This machine heats DNA to break it apart, then cools it so that the polymerase will attach to the DNA strands and make copies. For the heating process, the Open PCR machine is built with highly thermally conductive alloy of aluminum and insulation, allowing heat to be kept in the machine as well as spread the temperature across the block and be able to affect the test tubes. With a adjustable lid on top of the machine, condensation of water in the test tubes is essentially negated, allowing correct and reproducible results to appear. Last but not least, the Open PCR machine has the ability to connect to essentially any computer, compatible with a Windows or Mac though USB. Also the GUI program is only necessary for the initial and final steps, allowing a disconnection from the PC. The LED machine indicates the status of reaction, such as temperature, cycles, as well as remaining time until the reaction is complete. Finally, due to a file system interface via USB, the Open PCR machine can integrate into lab or other external control systems.

Experimenting With the Connections

When we unplugged (part 3) from (part 6), the machine's screen went black. The LCD screen did not receive power while the rest of the machine still did.

When we unplugged the white wire that connects (part 6) to (part 2), the machine did not read temperature properly. It briefly fluctuating upon detaching the wire, then read -40 degrees C.


Test Run

Wednesday, October 23, 2013
During our test run, we had some difficulty starting up the machine, as it had troubles starting up the process, and was constantly changing the time needed to create the first batch.




Protocols

Thermal Cycler Program
1. One cycle: 95 degrees Celsius for 3 minutes.
2. 35 cycles: Denature at 95 degrees Celsius for 30s, anneal at 57 degrees Celsius for 30s, extend at 72 degrees Celsius for 30s.
3. One cycle: 72 degrees Celsius for 3 minutes.
4. Hold: 4 degrees Celsius.


DNA Sample Set-up

Cancer DNA Template TUBE#: CON+ Positive Patient ID: 86763 TUBE#:1a ID:86763 TUBE#:1b ID:86763 TUBE#:1e
NON-Cancer DNA Template TUBE#: CON- Negative Patient ID: 65583 TUBE#:2a ID:65583 TUBE#:2b ID:65583 TUBE#:2e


DNA Sample Set-up Procedure

  1. First, the sample of DNA to be amplified is added.
  2. Both DNA primers are then added in succession.
  3. DNA Taq Polymerase is then added to the vials.
  4. Finally, loose nucleic acids are added so as to form the DNA copies.
  5. Place the tubes with the contents inside the Open PCR machine.
  6. Ensure that the lid is closed and everything is properly connected.
  7. Run Open PCR
  8. Gather tubes and use store them for use in the future.
  9. Clean up when done, ensuring all materials are accounted for.

PCR Reaction Mix

  • The PCR reaction mix is a combination of DNA to be amplified, two DNA primers, which allow for DNA Taq Polymerase to read and copy the desired sequence, DNA Taq Polymerase, for copying and reading the desired DNA, and nucleic acids, which are used to form the new copies of DNA.

DNA/ primer mix

  • Within the DNA/Primer mix is the sample of DNA with the desired strands to be replicated as well as RNA primer.





Research and Development

PCR - The Underlying Technology

THE COMPONENTS OF PCR
There are three steps in PCR, these steps are: 1)Denaturation, 2)Annealing, and 3)Extension. During the Denaturation, the DNA samples are heated to slightly under the boiling point of water, or around 94 degrees C. At this point the DNA strands break apart at the hydrogen bonds between the complementary bases, into the two basic strands that compose non-denatured DNA. During the annealing step, the DNA sample is cooled to around 54 degrees C where the forward and reverse primers attach on the single stranded DNA as the polymerase binds them, so as to allow the DNA Taq Polymerase to bind to the strands and create complementary copies. Finally, during the extension phase, the DNA is reheated to 72 degrees Farenheit, so as to stimulate the polymerase base pair copying reaction to build new DNA.





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