Name: Suhail Hiermandi Role: Research and Development, Machine Engineer
Name: Andrew Trujillo Role: Research and Development
Name: Elias Mloukhiyeh Role: Machine Engineer
Name: Emma Goddery Role: Experiment Protocol Planner
LAB 2 WRITE-UP
Thermal Cycler Engineering
Our re-design is based upon the Open PCR system originally designed by Josh Perfetto and Tito Jankowski.
System Design
Key Features
With the changes incorporated with the re-design of the Open PCR machine, the main design changes were to the LCD display screen, front side wall, and the motherboard. The changes would involve moving the location of the LCD display screen to the front side wall as well as adding buttons to the the wall that allow the Open PCR machine to be programmed instead of inputting the desired parameters into a computer with a USB cable. For the program to be on the machine itself, the motherboard would have to be re-programmed so that a computer connected to the Open PCR machine with a USB would no longer be needed to run the machine.
Instructions
The instructions on how to build the machine would be relatively the same as the before except for a few small changes. The front side wall would have to have a few more instructions to incorporate the LCD screen on it as well as the buttons used to program the machine. Besides that, the instructions to build the Open PCR machine would be the same as before.
Protocols
Materials
Cover to go over PCR machine || 1
Supplied in the Kit
Amount
Pipette Kit and Tips
One Micropipetter, 100 tips
PCR tubes
32
Primers, nucleotides, polymerase
One kit, can order refill packages
Instructions
1 booklet, code to download them online as well.
USB Cord
1
Power Cord (5 ft)
1
PCR machine
1
Adjustable Camera Stand to attach to PCR machine
1
Supplied by User
Amount
Screwdriver
1
Computer
n/a
DNA
n/a
Camera/Camera Phone/Webcam
1
PCR Protocol 1. Plug the PCR machine in and wait for the program to load completely. 2. On the LED screen on the top of the PCR machine, follow the steps to set up the cycles needed for the DNA replication. 3. Begin to set up the reactants by: -Put extracted DNA into a PCR tube.
-Add primers to the PCR tube.
-Add nucleotides to the PCR tube.
-Add DNA Taq Polymerase to the PCR tube.
-Place this DNA tube into DNA Thermal Cycler. 4. The cycle set up on the LED screen should be as follows: -Thermal cycler heats to 95 degrees Celsius for 30 seconds.
-Thermal cycler cools to 57 degrees Celsius for 30 seconds.
-Thermal cycler heats to 72 degrees Celsius for 30 seconds.
-These cycles happen 30 times.
DNA Measurement Protocol 1. Add 100 μl of SYBR green into each PCR tube being tested. 2. On the LED screen, activate the fluorescence cycle. 3. Set up the camera holder so that it goes under the PCR cover and is set up to view the tray of PCR tubes from the side. 4. Add a PCR tube with Calf Thymus DNA and SYBR green for a positive control and a PCR tube with only SYBR green for a negative control. 5. Put the PCR cover over the PCR machine. 6. Download the fluorescence camera app on a smartphone or computer with a webcam. 7. Put the camera or webcam in the camera holder and start the app's software to take pictures. 8. Images will directly download to the computer to be analyzed.
Research and Development
Background on Disease Markers
Rs33927012 is a cancer mutation in the DNA sequence that produces breast thyroid cancer. The mutation if found on the gene SDHB(6390), and the mutation is a change from a T to a C (TCT → CCT), in the sequence 5’-GAAGGATGAATCTCAGGAGG-3’. This cancer is most associated with Cowden-like syndrome. With this mutation symptoms are increased manganese superoxide dismutase function, and increased reactive oxygen species. This disease was found within two women with a wide age range, 29 and 54. What both patients had in common was that they had thyroid cancer, and both had a family history of breast cancer and papillary thyroid carcinoma.
A diseased allele will give the PCR a product because the primer that is given is specific for the disease. The primer will attach itself to the single stranded DNE at a sequence that matches the primer. If there is a replication as a product of a real time PCR run, then the DNA is positive for the cancer mutation and disease. If no replication takes place then the DNA is negative for the cancer mutation and disease.
BME 103 Fall 2012
