ISO setting: N/A (setting not able to be adjusted)
White Balance: N/A (setting not able to be adjusted)
Exposure: N/A (setting not able to be adjusted)
Saturation: N/A (setting not able to be adjusted)
Contrast: N/A (setting not able to be adjusted)
Calibration
Distance between the smart phone cradle and drop = 4 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 soluton (μg/mL)
5
80
80
2.5
2
80
80
1
1
80
80
0.5
0.5
80
80
0.25
0.25
80
80
0.125
0
80
80
0
Placing Samples onto the Fluorimeter
Put on gloves and lab coat
Set up camera and slide 4 cm away from each other
Make sure the camera is level with the very edge of the slide
Once camera is in place, place 80 μl of cyber green 1 onto the slide using the first two open spaces in the middle of the slide
Once the 80 μl of cyber green is in place, pipette 80 μl of the positive control into the cyber green.
Take 3 pictures of this mixture
Repeat steps 1-6 for the remaining DNA samples and the negative control
Data Analysis
Representative Images of Negative and Positive Samples
CALIBRATION
PART 1
PART 2
PCR REACTION
Image J Values for All Calibrator Samples
TABLE 1
TABLE2
Calibration curve
PCR Results Summary
Our positive control PCR result was 561 μg/mL
Our negative control PCR result was -357 μg/mL
Observed results
Patient 48611 :
Qualitative - The images looked like a bubble in the distance. Compared to the positive control the images were darker, and more similar to the negative control image.
Quantitative: These numbers are negative values, which is very obscure. This may have been a result from poor calibration.
Patient 56653 :
Qualitative: Much like patient 1, this patient had images similar to the negative control than the positive control.
Quantitative: These numbers are also very obscure, demonstrating negative concentration values.
Conclusions
Patient 48611 : This patient is more similar to the negative control than the positive control once the qualitative and quantitative values were taken into consideration.
Patient 56653 : Much like patient 48611, the data demonstrates that this patient is also closer to the negative control.
SNP Information & Primer Design
Background: About the Disease SNP
SNP, single nucleotide polymorphism is a DNA sequence variation occurring within a small population where a single base, A, T, C, or G differs between paired chromosomes or biological species. SNP are one of the most important genetic mutations that impact common disease. SNP results from replication errors and DNA damage, this phenomenon occurs exactly once in human evolution. Only sometimes does SNP have a correlation to a certain disease or trait. SNP has several applications in medicine such as gene discovery, allele mapping, and drug response prediction.
Primer Design and Testing
Results obtained from this lab were used to analyze the DNA sequence of two patients, one with a disease and the other without a disease. This lab demonstrated that primers bind to a certain region of a DNA sequence in order for the amplification of a small sample of DNA. Amplification occurs in two different directions on the DNA strand, the 5` and 3` ends. The 5` primers begins its sequence at the origin of the disease SNP variation location of 34370656. This was the location in the human genome where a thymine nucleotide has been mutated to a cytosine nucleotide, such a mutation results in the SNP disease that is seen in one of the patients. We used the UCSC In-Silico PCR website to test the non-disease primer to match the rs19956218 sequence.
BME 100 Spring 2015
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