Note to the grader - the info in this lab report was completed after the time deadline. No one in the group was aware that the lab report was due on April 7th at midnight. We have done our best to complete the report as soon as possible despite being late.
Procedure
Smart Phone Camera Settings
Type of Smartphone: iPhone 5s
Flash: Off
ISO setting: Default
White Balance: Auto
Exposure: Default
Saturation: Default
Contrast: Default
Calibration
Using the cradle provided, place the phone to be used onto the cradle. Make sure the camera is turned on. Then, position the camera at a distance where the camera is at the same height as the center of the drop. Doing so will provide the most accurate results.
Distance between the smart phone cradle and drop = 5.0 cm
Solutions Used for Calibration (Note: Only the Final DNA Concentration column values were used)
Initial Concentration of 2X Calf Thymus DNA (µg/mL)
Volume of 2X DNA solution (µL)
Volume of SYBR GREEN I Dye Solution (µL)
Final DNA Concentration (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
Wearing a pair of gloves, identify which side of the slide is “smooth”.
Turn on the fluorimeter.
Place the fluorimeter on the table.
Place the slide in the fluorimeter with the smooth side down.
Place the smartphone on the cradle.
Adjust the height of the fluorimeter or camera so that the camera can take a side-shot of the center of the drop.
Record the distance between the cradle and the fluorimeter light.
Using the micropipette, place an 80 µl drop of the SYBR GREEN I in the middle of the first two rows of the slide between two of the small hydrophilic dots.
Using a new micropipette tip, place an 80 µl drop of the first concentration of calf thymus DNA on top of the SYBR solution.
Move the slide so that the blue LED light is focused by the drop. It should hit the middle of the black fiber optic fitting on the other side of the drop.
Cover the fluorimeter as best as possible with the light box.
Take 3 independent images of the drop with the camera.
Remove the light box without moving the smartphone. (If the phone was moved, rearrange so that the distance matches the recorded distance)
Using the micropipette, remove the 160 µl drop from the slide. Dispose of the micropipette tip used.
Move the slide into the next clean hydrophilic dot position.
Repeat steps 8-15 for the remaining concentrations of calf thymus DNA.
Dispose of all materials responsibly and in their correct locations when done.
Data Analysis
Representative Images of Negative and Positive Samples
Positive Signal on ImageJ
Negative Signal on ImageJ
Image J Values for All Calibrator Samples (Values given in RAWINTDEN Drop - Background)
Final Concentration of DNA solution (ug/mL)
Image 1
Image 2
Image 3
Mean
St. Dev.
2.5
16684465
16066772
14173580
15641605.67
1308323.61
1
8078091
6669219
4855423
6534244.33
1615568.28
0.5
20016429
19470750
21676492
20387890.33
1148830.75
0.25
4444616
5071592
4658391
4724866.33
318730.22
0.125
3673678
4208643
4653738
4178686.33
490716.26
0
330973
228782
323620
294458.33
56996.07
Calibration curve
PCR Results Summary
Our positive control PCR result was 2.09 μg/mL
Our negative control PCR result was -8.50 μg/mL
Observed results
Patient 80565 : The drops turned bright green in color. The calculated concentration had a mean of 14.76 μg/mL for the three different primer mixes together.
Patient 87710 : The drops did not show any significant change. The calculated concentration had a mean of -5.11 μg/mL for the three different primer mixes together.
Conclusions
Patient 80565 : This patient had a greater concentration measured than the positive control. Therefore, the patient is positive.
Patient 87710 : This patient had a greater concentration measured than the negative control, but the value was still negative and much more so than the positive control. Therefore, this patient is negative.
SNP Information & Primer Design
Background: About the Disease SNP
The disease SNP under observation in this study is termed rs268, and it is a pathogenic SNP found on one of the alleles coding for lipoprotein lipase (LPL). It is found in humans at the 8:19956018 chromosome location, and the SNP involves a switch from an adenine nucleotide to a guanine nucleotide. The effect of this polymorphism on LPL is that it can disrupt the decomposition of lipoproteins. It can also lead to issues with protein binding and receptor binding. The possible results of these effects are diseases such as metabolic syndrome, coronary heart disease, and coronary artery disease.
Primer Design and Testing
After primers were designed for non-disease DNA, as well as SNP-carrying DNA, the primers were tested with the UCSC In-Silico Pro website. For the non-disease carrying forward and reverse primers, the result of the simulation was a 220 base pair DNA sequence matching a sequence from the non-disease LPL gene. For the disease carrying forward and reverse primers, no match was found even though the strand created from the non-disease primers differed by only one base pair. This shows that the disease primer designed is effective in only binding to the disease-carrying DNA, and not to non-carrying DNA. This makes it useful for testing for the presence of the SNP in patients.
BME 100 Spring 2015
