BME100 f2017:Group14 W1030 L5

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OUR TEAM

Name: Anshul Krishnan
Name: Lydia Gabric
Name: Sarah Bacon
Name: Charmayne Seaton
Name: Chris Ward


LAB 5 WRITE-UP

PCR Reaction Report

This lab dealt heavily with Polymerase Chain Reactions and the detection of pathogens. We use PCRs to multiply and amplify certain sequences of DNA. Since the PCR procedure is very complicated, it requires a number of supplies including Tag DNA Polymerase, a reaction buffer, template DNA and deoxyribonucleotides primers. For our lab, we were instructed to test for a pathogen in two different patients. After the template DNA, Tag Polymerase and primers were placed in a tube, they were mixed and placed into a thermocycler. This process separates the DNA and the primers duplicate the targeted segment of DNA. This entire process allows for the lab group to detect the specified pathogen in the two patients DNA.
Group 14 found that the prelab materials were very useful during this lab. We used the prelab readings as a basis to learn how to complete the micro pipetting for the replicate DNA of the two patients. A member of our group has had previous experience working in a similar lab situation, so we were able to differentiate between the first and second stop with little trouble. All of the final reactions had exactly the same amount of liquid due to careful pipetting. Additionally, at the completion of the lab there was no excess liquid in the DNA samples and PCR reaction mix. Since we took the time to label our tubes carefully and accurately during the first part of this lab, we did not have to revisit the labeling.

Fluorimeter Procedure

Imaging set-up
Our team first found a flat surface to place the camera and the box. We placed the iPhone 6 into the phone holder and leveled it using notebook paper. We staked our samples on different boxes we found to raise the samples to be leveled with the iPhone lense. The samples were raised to the correct height when we looked on the screen of the phone at eye level and saw the drop shape of the sample. When we captured our images we put the cover over so there would be a pure picture. After every picture we confirmed that each picture was focused and was not blurry.


Placing Samples onto the Fluorimeter

  1. Using gloves, find which side of the slide is the “smooth” glass back.
  2. Turn on fluorimeter and place it on table.
  3. Place a slide in fluorimeter with the smooth side down.
  4. Adjust height of fluorimeter (if needed) to get a camera view of the slide nearly edge on.
  5. Place 80 uL drop of SYBR Green I solution on first 2 clear circles in middle of slide.
  6. Place 80 uL drop of sample/calibration solution on top of the SYBR Green I drop.
  7. Adjust the slide so that the light illuminates the center of the drop.
  8. Cover with lightbox, but keep one flap up.
  9. Make sure droplet is in focus.
  10. As you put the flap down take picture.
  11. Remove the drop from slide and discard liquid in waste liquid container.
  12. Move the slide to the next position (center of next two circles).


Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA
(1) 5 μg/mL sample
BME100 f2017G17Lab5 5 ugpermL sample.PNG
(2) 0.5 μg/mL sample
BME100 f2017G17Lab5 0.5 ugpermL sample.PNG
(3) zero DNA
BME100 f2017G17Lab5 0 ugpermL sample.PNG
Calibrator Mean Values

Initial Concentration of 2X Calf Thymus DNA solution Final DNA concentration in SYBR Green I solution (µg/mL) Sample Number RAWINTDEN DROP - BACKGROUND MEAN Standard Deviation
(µg/mL)
Image 1 Image 2 Image 3
5 2.5 C-1 8722721 8726700 841261 6096894 4551512.126
2 1 C-2 5582815 5560250 5304232 5482432.333 154737.8878
1 0.5 C-3 5473255 5469271 5382837 5441787.667 51091.6225
0.5 0.25 C-4 3792077 3858805 3594819 3748567 137266.1361
0.25 0.125 C-5 2986747 3150828 3174538 3104037.667 102266.1547
0 0 C-6 2162654 2179780 2086915 2143116.333 49419.3016


Calibration curves

BME100 f2017G17Lab5Dot Plot 1-Callibration Curve.PNG

BME100 f2017G17Lab5Dot Plot 2-Callibration Curve.PNG
Images of Our PCR Negative and Positive Controls
(1) Negative Control
BME100 f2017G17Lab5 negativecontrol sample.PNG
(2) Positive Control
BME100 f2017G17Lab5 positivecontrol sample.PNG


PCR Results: PCR concentrations solved

PCR Product TUBE LABEL MEAN (of RAWINTDEN DROP - BACKGROUND) PCR Product Concentration (µg /mL) Total Dilution Initial PCR Product Concentration
(Step 5 calculation) (µg /mL)
(Step 6 calculation)
G14 + 5580913.333 487987.4186 12 5855849.023
G14 - 1730035 151271.0371 12 1815252.445
G14 1-1 1926789.333 168475.0101 12 2021700.121
G14 1-2 3874476 338778.4968 12 4065341.961
G14 1-3 1578819.333 138048.9127 12 1656586.952
G14 2-1 2346009.333 205131.1257 12 2461573.509
G14 2-2 1498387 131016.0018 12 1572192.022
G14 2-3 1252024.333 109474.333 12 1313691.996


PCR Results: Summary

  • Our positive control PCR result was 5855849.023 μg/mL
  • Our negative control PCR result was 1815252.44 μg/mL


Observed results

  • Patient 28550: The droplets of the first and third trials remained clear showing no green dye. However, the second trial’s droplet fluoresced a green color. The initial PCR product concentrations were 2021700.1 µg /mL, 4065342 µg /mL, 1656587 µg /mL
  • Patient 55154: The droplet remained clear and did not indicate any green dye. The initial PCR product concentrations were 2461573.5 µg /mL, 1572192.02 µg /mL, 1313692 µg /mL


Conclusions

  • Patient 28550:The patient 28550 had two droplets remain clear and one that illuminated. Since the droplets remained clear more than it illuminated, it can be concluded this patient tested negative.
  • Patient 55154: The patient 55154 had all its’ droplets remain clear. It can be concluded that this patient tested negative.