BME100 f2013:W900 Group3 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: Marissa KulickOpen PCR Machine Testing
Name: Marissa Kulick
Open PCR Machine Testing
Name: Blake WoodsProtocol Planning
Name: Blake Woods
Protocol Planning
Name: Shaun WoottenResearch and Development
Name: Shaun Wootten
Research and Development
Name: Bryce GonzalesResearch and Development
Name: Bryce Gonzales
Research and Development

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design
Image:OpenPCRImageFromOpenPCRwebsite.png

Source: http://OpenPCR.org

The Open PCR machine (left) is an inexpensive thermocycler for DNA replication. A thermocycler is a device that heats and cools a sample set to specific temperatures for a specific amount of time, in a specific sequence. This allows for repeated automated doubling of the DNA sample, freeing the researcher to attend to other responsibilities. As shown, it plugs directly into a computer via USB to allow the monitoring of the process in real time.


Experimenting With the Connections

Image:Openpcrmachineopen.png
Source: http://OpenPCR.org

When we unplugged the LED screen(part 3) from the circuit board (part 6), the machine's LCD screen turned off, though the machine seemed to still be running. It is unknown if the machine would operate this way, given that the LCD readout seemed to duplicate the information provided via USB connection to a computer.

Image:16wellPCRtubeblock.png
Source: http://OpenPCR.org

Image:Heated_Lid.png‎
Source: http://OpenPCR.org

When we unplugged the white wire that connects the circuit board(part 6) to the 16 well aluminum block (part 2), the machine stopped displaying the temperature readout. First, it displayed wildly variant temperatures for a few seconds, and then stopped functioning altogether. This would inhibit the function of the thermocycler as it needs to know how much heat to apply to the sample system to achieve desired denaturing/ annealing.


Test Run

The first test run occurred on 10/23 and the machine ran smoothly. The machine was surprisingly quiet for the amount of heat that needed to be dissipated. The only cause for concern that was noted was the lack of continuity on the part of the timing mechanism. The countdown feature seemed to fluctuate somewhat and we ended up stopping the program prematurely. Because we ran empty PCR tubes, it is unknown if the OpenPCR machine performed it's primary directive.




Protocols

Thermal Cycler Program


DNA Sample Set-up

Positive Control: Cancer DNA Template Patient 1 (ID 73190) Replicate 1 Patient 1 (ID 73190) Replicate 2 Patient 1 (ID 73190) Replicate 3
Tube label: CNC Tube Label: 101 Tube Label: 102 Tube Label: 103
Negative Control: Non-Cancer DNA Template Patient 2 (ID 43566) Replicate 1 Patient 2 (ID 43566) Replicate 2 Patient 2 (ID 43566) Replicate 3
Tube label: CNC Tube Label: 201 Tube Label: 202 Tube Label: 203


DNA Sample Set-up Procedure

  1. Step 1. Plug the OpenPCR machine into the power outlet and a computer and turn it on.
  2. Step 2. Pipette the 8 DNA samples into 8 differently labeled PCR test tubes using different pipette tips for each sample.
  3. Step 3. Pipette the primer mix into the 8 PCR test tubes.
  4. Step 4. Pipette the reaction mix into the 8 PCR test tubes.
  5. Step 5. Place the PCR test tubes into the OpenPCR machine.
  6. Step 6. Start the OpenPCR machine.
  7. Step 7. After machine has completed it's program, store the samples for use in a future lab.


Image:OpenPcrProgram.PNG
The settings should be changed to match those above.

PCR Reaction Mix

50 μL each
Taq DNA polymerase - the protein that is responsible for assembly of the newly created strand.
MgCl2 - a required co-factor for DNA polymerase
dNTP’s - the building blocks that will comprise the newly synthesized complimentary strand.

DNA/ primer mix

50 μL each
DNA template (different for each trial)
Forward primers (same for each trial)
Reverse primers (same for each trial)





Research and Development

PCR - The Underlying Technology


Template DNA is used as an example for the polymerase to create a complementary strand that lets the designed primers bind and without template DNA the generation of new DNA strands would be nonexistent. Primers are short pieces of nucleic acids that bind to the Template DNA after separation through complementary base pairing. Primers are predesigned to bind only to specific DNA sequences like the cancerous sequences that we are trying to duplicate. Primers come in both forward and reverse to connect to both sides of the DNA because DNA consists of two strands a leading and a lagging strand, naturally. TAQ polymerase is a protein enzyme that binds to primer locations and adds complementary nucleotides to the existing DNA strands generating a new sequence of DNA using the original template DNA that we started with. The TAQ polymerase also collects all the dNTPs to generate the desired sequence of cancerous DNA only. Magnesium Chloride (MgCl_2) works as a catalyst, because TAQ polymerase is a magnesium dependent enzyme. Deoxyribonucleotides (dNTP's) comprise the heavily synthesized strands. In other words, dNTP is what the TAQ polymerase adds.


The initial step takes place for three minutes at 95 degrees Celsius. During this step, the DNA polymerase is activated by heat. The denature step takes place at 95 degrees Celsius for another 30 seconds. This step is to guarantee that the hydrogen bonds in between the DNA absorb enough energy from the heating so the bonds break and single strands of DNA unzip from each other forming in the process. Next, the annealing step is held at 57 degrees Celsius for 30 seconds to allow the forward and reverse primers to bond to both sides of the single DNA strands. After annealing, the TAQ polymerase adds nucleotides to single DNA strands during the extension step with the magnesium chloride working as a catalyst for the extension, which takes place for 30 seconds at 72 degrees Celsius. Then, the final step occurs at 72 degrees Celsius for another three minutes. During this time, any remaining single strand DNA is extended. The final hold is held at 4 degrees Celsius to ensure that the new DNA strands do not degenerate, or separate and completely duplicate from the desired template DNA used. This process is then cycled through 35 times for enough of the sample cancerous DNA sequences to be formed.


The nucelotide Adenine (A) sticks to Thymine (T), and Thymine (T) also sticks to Adenine (A). Likewise, Cytosine (C) sticks to Guanine (G) the same way that Guanine (G) sticks to Cytosine (C).

Powerpoint illustrating how primers bind to the cancer DNA template, and how Taq polymerases amplify the DNA.
Media: Polymerase_Chain_Reaction.pptx





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