Name: Hope Haddad Role: Research and Development Specialist
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
Our redesign consisted of improving the insulation in the OpenPCR machine between the processing unit and fan and the the heating cap. This change is especially useful, allowing for increased insulation, with material such as fiberglass. This increased insulation allows for a more constant temperature at the heating cap. Because the temperature at the heating cap will be more constant, the OpenPCR machine will be able to perform its job faster and more efficiently, which is an incredibly handy feature considering a normal PCR machine can take up to two hours to amplify DNA. However, this increased insulation will cause the machine to become increasingly hot as it does its job. Our redesign accounts for this problem. By improving the fan on the machine as well, we eliminate any potential problems due to heat, such as overheating. However, another problem that arises from these new additions to the machine is room. The machine itself is fairly small, and compromising this portable size is not an option. For that reason, the redesign includes the use of a smaller fan, maintaining the intended efficiency of the redesign while accounting for the possible compromise in size. This redesign helps to improve the time that it takes for the OpenPCR machine to amplify DNA while maintaining a similar, if not smaller, size, allowing for much more DNA to be amplified in the same amount of time.
Key Features
-Improved fiberglass insulation
-Efficient smaller fan
-Faster DNA amplification
Instructions
The Open PCR machine is fairly easy to use. Simply plug it in to the USB port on your computer and turn it on. From your computer, you can adjust the temperature, add or remove steps to the process, and adjust the number of cycles. Even though the inside of the machine was redesigned, the instructions on how to use the machine have not changed.
Protocols
Materials
Supplied in the Kit
Amount
PCR Machine
1: $400- $600
Fluorimeter (LED glass stand)
1: $ 200.00
Connection USB cord for Computer to PCR 1
1: $20.00
Smartphone Stand
1: $ 5.00
Box
1: $ 5.00
Disk for ImageJ
1: $ 0.50
Improved Insulation
1: ~ $ 200.00
Efficient Smaller Fan
1: $ 50.00
Supplied by User
Amount
Computer
1: $ 1000
Pipettes
16 : $ 0.25 (16X)
Glass Slides
2 : $1.00 (2X)
Smartphone
1: $ 150.00
Small Tubes
32: $ 4.00
Water
Varies
DNA Samples
Varies
Reagent
Volume
Template DNA ( 20 nanograms)
0.2 microliters
10 micrometers forward primer
1.0 microliters
10 micrometers reverse primer
1.0 microliters
GoTaq Master Mix
50.0 microliters
Distilled Water
47.8 microliters
Total Volume
100.0 microliters
PCR Protocol
Setting up your Tubes
Collect 8 samples of DNA and 1 DNA(calf thymus standard at 2 micrograms/mL) sample and water from the scintillation vial to analyze.
With a permanent marker, number the transfer pipettes at the bulb so that you limit cross-contamination and only use ONE per solution, and number your Eppendrof tubes at the top. At the end, you should have 10 Eppendorf tubes and 10 pipettes clearly labeled.
Transfer each sample separately ( using one pipette per sample) into an Eppendorf tube containing 400 mL of buffer. Label this tube with the number of your sample. Make sure to transfer all of the sample into the Eppendorf tube. Use the same pipette to place ONLY this sample's drop onto the fluorescent measuring device.
Take the specially labeled Eppendorf tube containing the SYBR GREEN I and using it's assigned pipette, place two drops on the first two centered drops.
Now take the diluted sample and place two drops on top of the SYBR GREEN I solution drops.
Align the light going through the drop.
Take pictures with the smartphone, as many as desired.
If you are not satisfied with that sample, you may rerun that sample again or move on to the next sample.
Be sure to only run 5 samples per slide.
Before completing the lab, run the water from the scintillation vial as a BLANK using the same procedure listed above.
Smartphone Settings
Turn off the flash
Set ISO to 800
Set white balance to auto
Set exposure to the highest setting
Set saturation to the highest setting
Set contrast to lowest setting
PCR Machine
Acquire the DNA samples that have been submitted for testing
Insert Reactants into respective PCR tubes
Connect the PCR machine to the computer and press 'Start' to record data
Run the DNA samples through the thermal cycler program (see Stage 1)
Stage 1 (Initiation): 1 cycle at 95°C for 3 minutes, to separate the DNA strand.
Stage 2 (Denaturation): 35 cycles: first at 95°C for 30 seconds, and then gradually decrease the temperature to approximately 57°C for 30 seconds, and then raise the temperature to approximately 72°C for 30 seconds, so the DNA polymerase can be activated. This is also an example of heat shock, and is effective to initiate the addition of complementary nucleotides onto the DNA strand, which the DNA polymerase does.
Stage 3 (Elongation) : At this step, hold the DNA at 72°C for 3 minutes.The temperature is held here so that the DNA polymerase can copy the strand. Also, this is where the two desired fragments begin to appear- two strands that begin with primer one and end with primer two- and these are the DNA copies of the segment of DNA you began with.
Stage 4: Final hold until the DNA stabilizes at 4°C. At the end of this cycle you will have 8 fragments of the DNA
After the DNA has been through the numerous cycles, you will have over thousands of fragments of the same DNA sequences. After the DNA has been through the thermal cycle, mix each DNA sample with the PCR reaction mix (Taq DNA polymerase, MgCl2, dNTP’s, and a forward and reverse primer), using a separate pipette each time to reduce cross-contamination into 8 separate tubes
Image Processing
Upload pictures to ImageJ
Click on 'Analyze', choose 'Set Measurements', then 'Area Integrate Density', and 'Mean Gray Value'
Click 'Image', choose 'Color', and then 'Split Channels' to create three files
Choose image name that has 'green'
Select the 'Oval Selection' on the Menu Bar.
Click and stretch oval around green or clear drop in image. Click 'Analyze' and choose 'Measure'
Record sample number and measurements
Draw another oval of the same size in the 'green' file for the background above the drop to get the 'noise.' Click 'analyze' and choose 'measure'
Record sample name and measurements and use as background label
Save measurements to Excel file.
DNA Measurement Protocol
Research and Development
Background on Disease Markers
Tay-Sachs is a neural disease passed down from genetics. It can affect all age groups from infants to adults. You may be a carrier, but only two recessive traits will show symptoms of Tay-Sachs disease. The disease is caused by a defective gene on the 15th chromosome. Most signs are seen early on and can the disease can be tested for before birth. The affects of Tay-Sachs disease tend to work quickly. Some symptoms include loss of muscle function and strength, seizures, and/or blindness. Tay-Sachs disease in infants can kill a child by the age of four.
Tay-Sachs disease is caused by a mutation of HEXA alleles on the 15th chromosome. People with the disease have a low or nonexistent B-hexo enzyme, allowing buildup of the GMD2 ganglioslide lipid.
BME 103 Fall 2012
