LAB 5 WRITE-UP
Smart Phone Camera Settings
- Type of Smartphone: iPhone
- Flash: Inactive
- ISO setting: 800
- White Balance: Auto
- Exposure: Highest setting
- Saturation: Highest setting
- Contrast: Lowest setting
- Turn on the Blue LED excitation light.
- Turn on the smart phone camera and adjust the camera setting as listed above.
- Place the smart phone in a cradle of the Fluorimeter so a picture of the side of the drop will be taken.
- Adjust the camera to a distance where the first two rows of the slide will be as close as possible for a clear picture of the drop.
- Record the distance between the smart phone cradle and the drop using a ruler.
- Distance between the smart phone cradle and drop = 9.25 cm
Solutions Used for Calibration
| Initial Concentration of 2X Calf Thymus DNA solution (micrograms/mL)
|| Volume of the 2X DNA solution (μL)
|| Volume of the SYBR GREEN I Dye solution (μL)
|| Final DNA concentration in SYBR Green I solution (μg/mL)
Placing Samples onto the Fluorimeter
- Pipette a 80 μL drop of SYBR GREEN I in the middle of the first two rows of the slide, then add 80 μL of the calf thymus solution.
- Position the drop by moving the slide so the blue LED light is focused to the middle of the black fiber optic fitting on the other side of the drop.
- Set the camera to a 3 second timer to take a picture of the drop.
- Take three focused pictures of the drop.
- Pipette the 160 μL drop from the surface of the slide and move the slide to the next testing position.
- Repeat steps 1-5 for each trial.
Representative Images of Negative and Positive Samples
With DNA (Positive Signal)
Image J Values for All Calibrator Samples
PCR Results Summary
- Our positive control PCR result was 0.1151655833 μg/mL
- Our negative control PCR result was 0.114970375 μg/mL
- Patient 27468 : The drop was blue, and no green color appeared in the picture. The average value of the initial PCR product concentration for patient one was -0.01763836111 μg/mL.
- Patient 35294 : The drop was blue, and no green color appeared in the picture. The average value of the initial PCR product concentration for patient one was -0.07353509722 μg/mL.
- Patient 27468 : Inconclusive, because the first trial showed a positive result, while the remaining trials showed a negative result.
- Patient 35294 : Negative, because all of the trials showed a negative result.
SNP Information & Primer Design
Background: About the Disease SNP
SNP is a DNA sequence associated with disease. Mostly, SNPs occur within two alleles. SNP density can fluctuate depending on rate of mutation and genomic recombination. Furthermore, when a single nucleotide mutation occurs in a biological species, a SNP will occur. Also, SNPs mostly occur within non-coding regions rather than coding regions.
Primer Design and Testing
The non-disease primer (shown below) yielded results, because it did not include the SNPs.
The disease primer (shown below) did not yield results, because it included the SNPs.