Name: Alexandra Brunelle Research and Development Specialist
Name: Charul Singh Experimental Protocol Planner
Name: Alex Hoffmann Experimental Protocol Planner
Name: Student Role(s)
Name: Haley Gjertsen 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
Key Features
A. Now designed to be on the side of the lid
B. Glass oven like on the side
C. double layered to fit twice the amount of samples (32 samples)
New area for samples opens like an oven where the samples can be slid in. It will hold 32 samples instead of its original, 16. The sample trays will be placed one on top of an other when slid in. The glass is for seeing how tightly closed the oven lid is screwed on.
Instructions
1. Unscrew glass "oven-like door"
2. Slide 16 samples onto the top shelf and 16 samples into the bottom shelf
3. Close door
4. Screw the door tight but not until it touches the sides of the sample ependorf tube
5. Run PCR as original
Protocols
Materials
PCR Protocol Set up PCR Proceedures:
1. Label each tube differently and record label.
2. Insert reactants into their respective PCR tubes.
3. Connect assembled PCR machine to a computer.
4. Customize settings in program to two stages:
Stage 1: 1 cycle, heat to 95 degrees Celsius for 3 minutes
Stage 2: 35 cycles, heat to 95 degrees Celsius for 30 seconds; cool to 57 degrees Celsius for 30 seconds
Stage 3: heat to 72 degrees Celsius for 30 seconds.
5. Start up the OpenPCR program previously installed on the computer.
6. Slide out the tray that you want to place your tubes in.
7. Place tubes in the slots. and close lid tightly.
7. Look through the glass on the side to lower the top of the lid to make sure it touches the top of the tubes.
8. Press "Start" to run the PCR machine.
9. Record data.
Setting up the smart phone camera
Set the smart phone camera menu to the following:
1. Inactivate the flash.
2. Set ISO to 800.
3. Set white balance to auto.
4. Set exposure to highest setting.
5. Set saturation to the highest setting.
6. Set contrast to lowest setting.
DNA Measurement Protocol
1. Label each transfer pipette and Eppendrof tube to match the eight samples.
2. Pipette and Eppendorf tube for SYBR Green should be labeled with blue; DNA Calf Thymus Pipette and Eppendorf tube should be labeled with red; Label waste pipette.
3. Transfer each sample one at a time into the Eppendorf tube containing 400 mL of buffer with a matching label, using its respective pipette.
4. Take caution to not let pipettes touch each other.
5. Place two drops of SYBR Green (using the pipette with blue dot) onto the first two centered spots on the glass slide.
6. Place two drops of first diluted sample on top of the two drops of SYBR Green.
7. Turn on light and adjust glass slide so light goes directly through the drop.
8. With all equipment in the fluorimeter, Smartphone Operator will take pictures focused on the drop and give to image processor.
9. Using previously labeled waste pipette, remove drop from glass slide and discard waste.
10. Repeat steps 5 through 9 for each sample, using next two consecutive, centered spots on glass slide for next sample.
Image Processing
1. Upload photo to ImageJ.
2. Click on ANALYZE, choose SET MEASUREMENTS, then AREA INTEGRATED DENSITY and MEAN GREY VALUE.
3. Click IMAGE, choose COLOR then SPLIT CHANNELS to create three files.
4. Choose image name that has "green" next to it.
5. Active Oval Selection by clicking on Menu bar.
6. Click and stretch oval around green or clear drop in image. Click ANALYZE and choose MEASURE.
7. Record sample number and measurements.
8. Draw another oval of the same size in the "green" file for the background above the drop to get the “noise”. Again, click ANALYZE and choose MEASURE.
9. Record sample name and measurements and use as background label.
10. Save measurements to Excel file.
Research and Development
Background on Disease Markers Transient Myeloproliferative Disorder of Down Syndrome is a combination of the diseases leukemia, down syndrome and acute myeloid. It is extremely rare and takes the life of a patient in the very early months of life. It is associated with the SNP (single nucleotide polymorphism) 121912500 on chromosome 21. http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=121912500
Primer Design Transient Myeloproliferative Diorder of Down Syndrome: The forward primer of this disease is ACCGGCTGGTGGGCCCGCTG. The reverse primer that would correctly match the forward primer and not code for a diseased patient would be TGGCCGACCACCCGGGCGAC. The diseased allele and diseased patient will have a reverse primer TGGCCGACAACCCGGGCGAC. Therefore, a patient that contains the disease has an allele mutation within the pairing of G to C, it is instead G to A. This will give a positive on the PCR machine because it becomes single stranded due to the fact it cannot replicate properly. This, when we apply the SYBR green it will attach to only the single primers and the diseased patients will glow green. The replicated, normal strands, will appear clear when the SYBR green in added because it will not be able to attach to the replicated strands.
Illustration
Below is a visual representation of what happens during a Polymerase Chain Reaction:
1. The DNA is heated up to 95 degrees Celsius, splitting the strand apart. 2.The solution is cooled down to 50 degrees Celsius and primers are able to attach to the separated strands. 3.The solution is heated to 72 degrees Celsius and taq Polymerase attaches. 4. The DNA strands are replicated at 72 degrees Celsius. 5. The process repeats as the PCR machine heats up to 95 degrees.
(Photo Credit: The images used here were borrowed from OpenPCR.) Bonus:
Bayesian statistics allows you to analyze and use all available data and can help you under the limitations of diagnostic tests. In our case, it can be used to test a sample of patients for this disease. From the tests we can see the false positive rate, sensitivity the test has to the disease, and prevalence to better inform the public on the statistics of the disease within the population and the subject to failure the test has so the doctors can run more if necessary.
D=disease positive
T=positive test
p=probability
p(DIT)=[p(TID)*p(D)]/[(p(TID)*p(D))+(p(TI~D)*p(~D))]
BME 103 Fall 2012
