Our re-design is based upon the Open PCR system originally designed by Josh Perfetto and Tito Jankowski.
System Design
The old design of the PCR Machine is as follows, including only 16 tube slots:
The goal of our PCR redesign is to add more test tube space, making the PCR more efficient:
In order to increase tube space, we increase the heating plate and increase the power proportionally to the size of the plate. We would also add more ventilation next to the heating plate in order to speed up the cooling process of the DNA.
Key Features
Critical features to our design include an increase in tube space from 16 tube slots to 96. In doing this, the area of the heating plate is increased relative to the space the tubes take up. The increased area of the heating plate will require added ventilation to speed up the cooling process. The increased heating plate is necessary, because the PCR requires a series of heating up and cooling down the DNA to specific temperatures so that the DNA will multiply.
The benefits of increasing the tube space would be that the PCR would be more efficient. It would be more efficient because the Machine would be able to test more subjects at the same rate, allowing for increased amount of trials and increased amount of patients.
Instructions
Protocols
Materials
Supplied in the Kit
Amount
SYBR Green I
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Calf Thymus DNA
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PCR tubes
95 tubes
Negative control
1
Positive control
1
Pipettes
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Microtubes
93 tubes
GoTAQ Master Mix
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Fluorimeter
1
Fluroimeter stand and box
1
CD with ImageJ software
1
Supplied by User
Amount
DNA samples
93 samples
Smartphone for fluorimeter
1
External computer
1
Heatproof marker pen
1
PCR Protocol
First, DNA samples were obtained from 31 different patients, each giving 3 DNA samples each. These DNA samples were stored in a freezer in microtubes. For patient number 1, his or her DNA samples were labelled 1a, 1b and 1c, using the heatproof marker. The other DNA samples from the remaining 30 patient were labelled in the same way. After all the DNA samples were obtained, each sample was transferred over to PCR tubes, which are tubes that are used especially for the PCR process. Each PCR tube was labelled in the same way as before. In addition to these 93 PCR tubes, two more tubes, each containing a negative control and a positive control respectively. The PCR tube containing the negative control was labelled "-" and the PCR tube containing the positive control was labelled "+". Using separate pipettes for each DNA sample and the controls, Primer 1 was added to every PCR tube. Once Primer 1 is added, Primer 2 is added to all the tubes as well, with separate pipettes still being used for all transfers. AFter these two primers are added, nucleotides are added in the same way. Finally, DNA Polymerase is added to all the PCR tubes.
All 95 of the PCR tubes are placed into the PCR machine, and the software for the PCR machine was opened on the computer that the PCR machine was connected to.
DNA Measurement Protocol
The fluorimeter was set up according to the image shown below.
Research and Development
Cystic Fibrosis is a genetically inherited disease that is derived from the production of excessive mucous build up in the respiratory tract. This has a major effect on the body's endocrine system, more specifically in the digestive and respiratory systems.
Background on Disease Markers
The marker SNP for the disease of cystic fibrosis that is being used is rs35731153. This disease can be found on chromosome 16. Cystic fibrosis gene is a mutation in the gene for the protein cystic fibrosis transmembrane conductance regulator. The cystic fibrosis gene mainly affects the lungs but also the pancreas, liver, and intestines. It is a scarring and cyst formation in the pancreas and causes trouble breathing. This often leads to lung infections. Ultimately death occurs unless a lung transplant can be completed.
BME 103 Fall 2012
