BME100 s2014:T 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

Mikaela HallRole: Research and Development
Mikaela Hall
Role: Research and Development
Sarah McBryanRole: Research and Development
Sarah McBryan
Role: Research and Development
Avery WittingRole: Machine Engineer
Avery Witting
Role: Machine Engineer
Daniel SaundersRole: Protocol Planner
Daniel Saunders
Role: Protocol Planner

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design

OpenPCR Machine and Software OpenPCR External Components OpenPCR Internal Components
Original image.
Original image.

Labels added. Image source: http://openpcr.org/v1story
Labels added. Image source: http://openpcr.org/v1story
Labels added. Image source: http://openpcr.org/v1story
Labels added. Image source: http://openpcr.org/v1story

The first image shows the initial set-up between the PCR machine and the MacBook that was used to collect the readings and data. The computer interface allowed the group to set the temperature for each cycle, the duration of each cycle, and the number of cycles to be run. It used the PCR program to run the machine. The PCR machine interface provided details on what cycle/step the machine was on at any given time.

The second image shows a close up of the PCR machine. Labels show the handle, used to open and close the machine, as well as the heating lid which raises the temperature of the PCR for the specified cycles.

The last image shows the inside layout of the PCR machine. Labels show the power supply, LCD screen, fan, heat sink, and the brains board. The power supply, as the name suggests, gives the machine the power necessary to run the PCR. The LCD screen is the machine interface which shows what is happening in the machine. The fan provides the cooling for the necessary cycles. The heat sink pulls the heat away from the aluminum block. The brains board is the motherboard that connects to the computer, giving the machine directions on what to do.


Experimenting With the Connections

When we unplugged the screen from the motherboard, the machine's screen turned off.

When we unplugged the white wire that connects the motherboard to the cooling plate, the machine's temperature sensor was no longer connected.


Test Run

We first tested the Open PCR on March 20. The machine operated as designed. It powered on and connected to a Macbook. The software installed and ran well. It displayed the same information that was displayed on the LED screen. We ran the Open PCR for 10 cycles. It completed the cycles without incident.

Protocols

Original image.
Original image.

Thermal Cycler Program

  1. Start Open PCR Software
  2. Select "Add a New Experiment"
  3. Click "More Options"
  4. Enter Program Parameters:
    • HEATED LID: 100°C
    • INITIAL STEP: 95°C for 2 minutes
    • NUMBER OF CYCLES: 35
    • Denature at 95°C for 30 seconds
    • Anneal at 57°C for 30 seconds
    • Extend at 72°C for 30 seconds
    • FINAL STEP: 72°C for 2 minutes
    • FINAL HOLD: 4°C


DNA Sample Set-up

Positive Control Patient 1: Replicate 1 Patient 1: Replicate 2 Patient 1: Replicate 3
Negative Control Patient 2: Replicate 1 Patient 2: Replicate 2 Patient 2: Replicate 3

Test Tube Lay-out

dD pf1 pf2 pf3
nD pm1 pm2 pm3


DNA Sample Set-up Procedure

  1. Set up materials, label all tubes
  2. Place the PCR mix into each of the eight labeled tubes
  3. Add the negative/positive controls to the designated first tube on each row
  4. Add the DNA from patient 1 (ID: 41731F) to tubes 2-4 in row one
  5. Add the DNA from patient 2 (ID: 78042M) to tubes 2-4 in row two
  6. Place all labeled tubes into the already tested PCR machine
  7. Make sure the samples run at least 20 cycles
  8. Use fluorescence app to test each sample


PCR Reaction Mix

  • PCR buffer
  • Taq DNA polymerase
  • Magnesium Chloride (MgCl2)
  • Nucleotides (dNTPs)


DNA/ primer mix

  • Template DNA
  • Disease-specific reverse and forward primers



Research and Development

PCR - Functions of Components
PCR, or a polymerase chain reaction, is a laboratory procedure in which a specific area of DNA is delineated by primers and amplified. During a standard PCR, a specific sequence will be synthesized into billions of copies. There are two main components of PCR made up of several different components: the PCR reaction mix and the DNA/primer mix. The reaction mix is made up of the PCR buffer, Magnesium Chloride, Taq DNA polymerase, and nucleotides or dNTPs. The primer mix is comprised of the template DNA, and the disease-specific reverse and forward primers.

The first part of this lab involves mixing all the components together in a small micro tube. They are all mixed in the PCR buffer. This buffer not only serves as the liquid environment in which the reactions can take place, but it also serves as a stabilizer which keeps pH levels, ionic strength, and co-factors constant. The magnesium chloride is one such co-factor. It helps to increase the activity of the magnesium dependent enzyme, in this case: Taq DNA polymerase. The Taq DNA polymerase, as stated earlier, is an enzyme. In a PCR, it serves as the synthesizer of the specified, or target, DNA sequence. The dNTPs serve as the building blocks for the polymerase. They provide the ingredients that the Taq DNA polymerase will use to build/synthesize the new copies of DNA.

The DNA/primer mix is then mixed into the solution PCR reaction mix. The first part of this is composed of the DNA template. This provides the target sequence that the PCR is designed to amplify. Without this, the reaction has nothing to synthesize. The second part of this primer mix is the forward and reverse primers. These primers are specified to find and latch onto specific parts of a DNA sequence. They serve as markers for the Taq DNA polymerase showing where to start copying a target sequence and where to stop.


PCR Steps
PCR reactions involve three steps (denature, anneal, and extend) that are all performed at different temperatures. The first step uses a high temperature, usually between 92-95°C in order to denature the double stranded DNA. This means that the two strands are separated from each other. During the second step, the temperature is lowered so that the primers can anneal, or bind, to the single stranded DNA. The optimal temperature will vary based upon the melting temperature of the primer-template hybrid, but it must not be too high or too low. Finally, the temperature is raised once more so the DNA polymerase can synthesize the DNA. For Taq polymerase, the polymerase used in this OpenPCR lab, the temperature during the third step should be around 72-78°C.


Base Pairing
Adenine (A) always pairs with thymine (T), while cytosine (C) always pairs with guanine (G). These pairs occur due to hydrogen bonding. Adenine and thymine establish two hydrogen bonds and cytosine and guanine establish three hydrogen bonds.

Original image.
Original image.




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