BME100 s2016:Group14 W1030AM L5

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OUR TEAM

Name: Jessica Kerlee
Name: Derek Vielhauer
Name: Tessaly Alexander
Name: Brent Kiracofe
Name: Bianca Garcia
Name: Talha Ghanchi


LAB 5 WRITE-UP

PCR Reaction Report

Since everyone in the group watched the tutorials or have done previously work with pipettes, the pipettes were not a problem to work with. Some of the group members needed a little review for the difference between the first and second stop for the pipettes. The first stop was pressed before having any liquid touching the pipette, which afterward, the pipette would be released, which would bring the liquid in to the pipette. The liquid was not the same in each of the final reactions, which made there be a slight leftover liquid. Since our group was actually organized, the labeling scheme that we used worked well.

Fluorimeter Procedure

Web camera set-up
Type of Smartphone:iPhone 6s

  • Flash:No Flash
  • ISO setting: 800
  • White Balance: No
  • Exposure: No
  • Saturation: No
  • Contrast: No

Placing Samples onto the Fluorimeter

  1. The calibration method used to begin this experiment is placing 160 μL drop of water in the middle of the first rows located on the slide using a pipettor
  2. The excitation light got the Blue LED light was turned on using the switch
  3. The camera on the smartphone being used was prepared and placed in the cradle at a right angle of the slide. Necessary adjustments were made in order to capture the best angle of the drop
  4. The cradle containing the phone was then adjusted so that the distance between it and the first two rows on the slide were as close as possible without the image becoming blurry or disturbed.
  5. The distance between the smartphone cradle and the drop were recording using the ruler provided, with lots of caution.
  6. An 80 μL drop of SYBR GREEN I was placed in the middle of the first two rows on the slide. Then 80 μL of one of the calf thymus (water blank) solutions were added. After this occurred, the sample on the slide was now considered a drop.
  7. The light box provided was placed over the fluorimeter and the camera, removing as much stray light as possible.
  8. Three images were taken using the smartphone, being careful and checking that the drop was focused in every image.
  9. Remove the light box as careful as possible without moving the smart phone.
  10. Remove the 160 μL drop from the surface using the pipettor and move the slide to the next position.
  11. Repeat steps 5-10 using different concentrations of calf thymus DNA


Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA
Description of image Description of image Description of image

Order of images left to right: 5 μg/mL, .5 μg/mL, and zero DNA

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 784214 810886 820724 805274.6667 18890.74698
2 1 C-2 720784 754188 765290 746754 23165.58387
1 0.5 C-3 720913 747526 753916 740785 17503.72283
0.5 0.25 C-4 721951 698932 686966 702616.3333 17781.12174
0.25 0.125 C-5 642089 626108 663498 643898.3333 18760.55144
0 0 C-6 728567 720941 659668 703058.6667 37770.37774


Calibration curves
Description of image

Description of image


Images of Our PCR Negative and Positive Controls
Description of image Description of image

From left to right: Positive and Negative controls


PCR Results: PCR concentrations solved

PCR Product TUBE LABEL MEAN (of RAWINTDEN DROP - BACKGROUND) PCR Product Concentration Total Dilution Initial PCR Product Concentration
Positive Control 2665317 78.85864062 12 946.3036875
Negative Control 1746504.33 42.23066893 12 506.7680271
1-1 2670103 79.04943193 12 948.5931832
1-2 2721205 81.08658561 12 973.0390273
1-3 2531403 73.52023121 12 882.2427746
2-1 1961057.33 50.78370859 12 609.4045031
2-2 1849168.67 46.32332749 12 555.8799298
2-3 1936383.33 49.80009288 12 597.6011146


PCR Results: Summary

  • Our positive control PCR result was 946.3036875 μg/mL
  • Our negative control PCR result was 506.7680271 μg/mL


Observed results

  • Patient 20710 :The images of this patient a dark green light from the photos taken, which was very similar to the positive droplets. These images showed luminescence inside of them. The observed concentration of the droplets from this patient had an average of 946.304 μg/mL.
  • Patient 35451 :The images of this patient were dark with very little if not any luminescence. They showed very little green light compared to the positive droplets. The observed concentration for this patient had an average of 506.7680 μg/mL.


Conclusions

  • Patient 20710 :While looking at the data analysis and the pictures, this patient appears to be similar to the positive control due the green light and it what somewhat luminescence. The patient had an average of 921.627 μg/mL, which is very close to the positive sample of 946.304 μg/mL. This means that the patients was probably positive.
  • Patient 35451 :When looking at the data, this patient appears to be similar to the negative control due to the lack of green light within each droplet. The average concentration of 587.629 μg/mL was very close to the concentration of the negative sample which was 506.768 μg/mL. Therefore, this patient was negative.