BME100 f2017:Group8 W1030 L5

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OUR TEAM

Name: John Carey
Name: Elizabeth White
Name: Jennifer Brodsky
Name: Jacob Hayes
Name: Kristin DeJesus


LAB 5 WRITE-UP

PCR Reaction Report

Micropipettes are used to measure and deliver accurate volumes of samples. The pre-lab reading was useful and thoroughly explained the process of using a micropipette, including the calibration as well as the first and second stops. The first stop is used to create a suction for the sample. Then, the second stop is used to dispense of the sample collected. The final reactions did have the same amounts of liquid (80 mL of SYBR Green + 80 mL of the DNA sample). When combining the DNA sample and PCR mix, there was no left liquid. The labeling scheme used did not need to be altered because accuracy was achieved.

Fluorimeter Procedure

Imaging set-up
To set up the fluorimeter for testing, a glass slide was inserted with the smooth side down, and rough hydrophobic side facing up. A smart phone (iPhone 6s) was then set up on a cradle roughly 5 centimeters (cm) away from the fluorimeter. Various camera settings were altered in order to capture the best image. The ISO was set to 800, and the exposure, as well as saturation, was set to the highest setting, while contrast was set to the lowest setting. White balance remained on auto. The fluorimeter was set on top of the box of 69 micropipette tips. A light box was placed around the fluorimeter to cancel exterior lighting. An image was then captured using the smart phone with a 3 second timer under the light box. Flash was inactivated. Although, the smartphone used had an automatic flash during the timer, the image captured was not affected.


Placing Samples onto the Fluorimeter

  1. The micropipette was set to 80 µl.
  2. Using a new micropipette tip, an 80 microliters (µl) drop of SYBR GREEN was placed on the rough side of the slide, in between two circles in a row. The micropipette tip was then discarded.
  3. Using a new micropipette tip, an 80 µl sized drop of sample (calf thymus or DNA) was added.
  4. The fluorimeter was turned on.
  5. The slide was adjusted so the blue LED light from the fluorimeter focused by the middle of the drop.
  6. The light box was then placed over the fluorimeter to cancel out room light.
  7. The smartphone cradle was placed 5 cm away from the fluorimeter.
  8. A timer on the camera of the smartphone was set to 3 seconds (sec).
  9. The capture image button was pressed, and the lid to the light box was shut to achieve complete darkness inside the box.
  10. Two additional images were captured of the drop on the fluorimeter.
  11. The above procedures were repeated for all calf thymus and DNA samples.


Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA

5 μg/mL sample 5b100(1).png


0.5 μg/mL sample .5b100.png


zero DNA Waterb100.png


Calibrator Mean Values

Initial Concentration of 2X Calf Thymus DNA solution (micrograms/mL) Final DNA concentration in SYBR Green I solution (µg/mL) Sample Number RAWINTDEN DROP - BACKGROUND ' ' MEAN Standard Deviation
Image 1 Image 2 Image 3
5 2.5 C-1 7686121 8137131 7576737 7799996.333 297045.5394
2 1 C-2 7887613 7529890 7510326 7642609.667 212404.4786
1 0.5 C-3 9040005 9743871 10174109 9652661.667 572527.1413
0.5 0.25 C-4 6323499 6262843 7187453 6591265 517203.9115
0.25 0.125 C-5 3662713 3996690 3618109 3759170.667 206903.2491
0 0 C-6 3217202 2873736 4647033 3579323.667 940475.7512


Calibration curves

Dot Plot 1!.png

Dot Plot 2!.png

Images of Our PCR Negative and Positive Controls

Negative control PCR sample G-b100.png


Positive control PCR sample G+b100.png


PCR Results: PCR concentrations solved


PCR Product TUBE LABEL MEAN (of RAWINTDEN DROP - BACKGROUND) "PCR Product Concentration (µg /mL)(Step 5 calculation)" Total Dilution '"Initial PCR Product Concentration (µg /mL)(Step 6 calculation)"
G8 + 8277744.667 1.571613744 12 18.85936492
G8 - 822826.667 0.00000007986669905 12 0.0000009584003886
G8 1-1 1132567233 3.475068409 12 4.170082091
G8 1-2 1263429.667 0.5866370255 12 7.039644306
G8 1-3 1081264 0.642688 12 7.712256
G8 2-1 5023961 0.5704495385 12 6.845394462
G8 2-2 11472760.33 2.554695486 12 30.65634583
G8 2-3 12767161 2.952972615 12 35.43567138


PCR Results: Summary

  • Our positive control PCR result was 18.859 μg/mL
  • Our negative control PCR result was 9.6E(-7) μg/mL


Observed results

  • Patient 28692: The droplet remained clear; no green was detected once the LED light was focused on the middle of the drop of DNA plus SYBR Green. The concentration of patient 28692 averaged to 7 μg/mL resulting in this patient testing negative.
  • Patient 68634: The droplet showed green under the PCR reaction; green was detected once the LED light was focused on the middle of the drop of DNA plus SYBR Green. The concentration of patient 68634 averaged to around 30 μg/mL, resulting in this patient testing positive. For the first trial, patient 68634's concentration was closer to the negative control. However, the remaining two trials resulted in concentrations closer to the positive control, resulting in an overall positive result.

Conclusions

  • Patient 28692: This patient tested negative for the disease single-nucleotide polymorphism (SNP).
  • Patient 68634: This patient tested positive for the disease single-nucleotide polymorphism (SNP).