BME100 f2014:Group10 L5

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BME 100 Fall 2014 Home
Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
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Name: Meredith Bothman
Name: Dawei Jiang
Name: Peter Seo
Name: Cera Lange
Name: Steven Nguyen



Smart Phone Camera Settings

  • Type of Smartphone: iPhone 5s
    • Flash: Off
    • ISO setting: Auto
    • White Balance: Auto
    • Exposure: Auto
    • Saturation: Auto
    • Contrast: Auto

The phone cradle should be 4 centimeters from the fluorimeter. The photo should be taken edge-on which means you should see the side of the plate and the drop. The photo needs to be taken with no light so it is suggested that you set a 3 second timer on the phone and turn off the flash. No light should enter the box while the picture is being taken.

  • Distance between the smart phone cradle and drop = 4 centimeters

Solutions Used for Calibration

Initial Concentration of 2X calf Thymus DNA solution (µg/mL) Volume of the 2X DNA solution (µL) Volume of the SYBR Green 1 Dye solution (µL) Final DNA concentration in SYBR Green 1 solution (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

  1. Use the switch for the Blue LED to activate the excitation light.
  2. Adjust the settings of your smartphone's camera and make sure the camera is on.
  3. Adjust the height of the fluorimeter with the plastic trays so your smartphone takes the photos of the drop sideways. Your smartphone should be in the cradle at a right angle from the slide.
  4. Adjust your smartphone so that is is about 4 centimeters away from the slide, or at close as you can get it to the slides without the image being blurry.
  5. Use a ruler to record the distance between your smartphone and the drop. Try not to move any of the components too much or else the light collected might show a significant difference between images.
  6. Place a 80 microliter drop of SYBR GREEN I between the first two rows of the slide. Then add a 80 microliter drop of a calf thymus solution. This is a sample.
  7. Adjust the slide so that the blue LED light is focused by the drop to the cener of the black fiber optic on the other side of the slide.
  8. Set the timer on the smartphone so that photos may be taken after covering the fluorimeter and the phone without letting in excess light.
  9. Make sure the drop is focused and take 3 photos.
  10. Remove the box without moving the smartphone.
  11. Take off the 160 microliter drop and readjust the slide to the next position.
  12. Repeat steps 6-11 for the other solutions of calf thymus DNA.

Data Analysis

Representative Images of Negative and Positive Samples

Negative Image This image represents a sample with no DNA present.

Positive Image This image represents a sample with DNA present.

Image J Values for All Calibrator Samples

Final DNA concentration Area Mean Pixel Value Raw Intden Drop Raw Intden Background Raw Intden Drop- Background
0 20028 53.351 1068506 50928 1017578
0 19320 55.489 1072042 54994 1017048
0 21484 58.681 1260713 58809 1201904
0.25 25960 102.281 2655209 77962 2577247
0.25 28664 102.309 2932581 85834 2846747
0.25 30910 95.699 2958067 59798 2898269
0.5 26840 115.051 3087973 131143 2956830
0.5 29768 112.369 3344991 81490 3263501
0.5 26076 111.946 2919104 60412 2858692
1 26544 140.603 3732158 103219 3628939
1 26504 146.017 3870039 125554 3744485
1 30572 139.678 4270228 137375 4132853
2 28500 188.764 5379771 69598 5310173
2 31372 186.659 5855870 70684 5785186
2 29300 189.379 5548799 67173 5481626
5 28960 231.246 6696891 70908 6625983
5 29034 224.763 6525315 71385 6453930
5 28600 231.416 6618501 85853 6532648

Calibration curve

Positive Image

PCR Results Summary

  • Our positive control PCR result was 2.931 μg/mL
  • Our negative control PCR result was 1.131 μg/mL

Observed results

  • Patient 14376 : The images for patient 14376 showed that the drops were very fluorescent. The drops glowed bright green, and appeared opaque. The DNA concentration for the samples was found to be 4.227 μg/mL.
  • Patient 62548 : The images for patient 62548 showed slight fluorescence. They did not show any signs of glowing, but the drops had a green hue and were more translucent than transparent. The DNA concentration for the samples was found to be 0.6797 μg/mL.


  • Patient 14376 : The patient tests positive for the disease because the DNA concentration of the samples, 4.227 μg/mL, is much higher than the DNA concentration of the positive control 1.131 μg/mL.

Note: Due to labeling errors, it was not possible to determine the patient IDs of the different samples. The most probable ID was assigned to each sample, but this could be inaccurate.

SNP Information & Primer Design

Background: About the Disease SNP

A disease SNP is made up of a nucleotide and a polymorphism and affects a specific allele sequence. A nucleotide is a structural component of DNA/RNA and consists of bases such as adenine, thymine, guanine, and cytosine. A nucleotide is a deoxyribose sugar group bonded with one of the four bases. A polymorphism is a genetic variation which is a difference in a DNA strand among individuals. Polymorphism is when two or more phenotypes (physical traits) exist in the same population of species. This may show variations in health among the individuals, and if more than one percent of a population is positive for the variation, it is useful for genetic linkage analysis. The mutation is a missense mutation where there is a single nucleotide change in the codon.

Primer Design and Testing

To find the non-disease forward, we counted 19 bases on the top row from the left including the T base. For the reverse primer, we searched for the 34370856 base (200 to the right from position 34370656) and then counted 19 to the left on the bottom row. For the disease forward primer, we swapped the T with C and that resulted in no results coming up in the search.

Non-disease Negative Image Disease Negative Image