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.
BME 103 Fall 2012
