BME100 f2015:Group4 1030amL5

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Contents

OUR TEAM

Name: Gabrielle Mills
Name: Gabrielle Mills
Name: Christa Deckman
Name: Christa Deckman
Name: Neil Rastogi
Name: Neil Rastogi
Name: Ngan Nguyen
Name: Ngan Nguyen
Name: Evan Higgs
Name: Evan Higgs
Name: Dylan Bunch
Name: Dylan Bunch


LAB 5 WRITE-UP

PCR Reaction Report

Our pipetting of the samples was successful during the setup of this laboratory procedure. The appropriate amounts of DNA sample liquids and PCR reaction mix were added to the tubes with no observable error. The tubes were labeled as we had previously planned in the earlier portion of this lab, and we successfully transferred the liquids between the tubes without contamination.The pre-lab provided appropriate information about how to complete a PCR lab. Through the interactive activities, we were able to familiarize ourselves with the important and significant procedure. In addition, the reading provided informative background descriptions and definitions that helped in the progression and completion of the laboratory procedure. The first and second stops on the pipettor were easy to distinguish after the first few pipetting steps. When you hit the first stop on the pipettor, it is quite difficult to go past it accidentally. Therefore, when you purposefully expel the sample into the PCR tube, you can perceive when it is fully expelled at the second stop. The final reactions held the same amount of liquid as the original components combined. Precision was used in transferring the components into the reaction, and therefore, there was the exact same amount of liquid. There were no component liquids left in the tubes of DNA and the PCR reaction mix. Since precision was used in removing these liquids, there was no liquid left in these tubes and all was added into the PCR tubes. We used our original labeling scheme from Part A for the procedure in Part C. We saw no need to change it, given that it was unique to our group seeing as we labeled our PCR tubes with “G4.”

Fluorimeter Procedure

Smart Phone Camera Settings

  • Type of Smartphone: HTC 1
    • Flash: off
    • ISO setting: 1600
    • White Balance: auto
    • Exposure: 2x
    • Saturation: 2x
    • Contrast: -2x

Camera set-up


The phone, with camera included, was placed in the phone cradle in front of the fluorimeter. Once the drop was set on the slide, the camera was shifted to a closer distance while retaining the focus on the drop.


  • Distance between the smart phone cradle and drop (beam on fluorimeter) = 7 centimeters


Placing Samples onto the Fluorimeter

  1. Place the smooth side of the slide downward
  2. After setting the camera timer to three seconds, place it in the provided cradle
  3. Change the height of the fluorimeter to get a side view of the drop on the camera
  4. In the middle of the slide, put an eighty microliter drop of SYBR Green 1 upon the first two clear circles.
  5. Then, put an eighty microliter drop of the sample on this drop.
  6. Shift the slide so that the light shines through the drop, focusing the light
  7. After adjusting the distance between the camera cradle and fluorimeter so that the images are close, yet focused, record this distance
  8. Place the lightbox over the set up with one flap open
  9. Set the timer and close the flap before it captures the image
  10. Place the drop in the waster container after recording which sample was just pictured
  11. Next, slide the slide into the next position and repeat the experiment for all the possible slide posititons


Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA

Image:PotatoJellyfish.jpg


High

Image:Gellyfish.jpg


Low

Image:DancingQueen.jpg


Zero

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: Image 1 RAWINTDEN DROP - BACKGROUND: Image 2 RAWINTDEN DROP - BACKGROUND: Image 3 Mean Standard Deviation
5 2.5 C-1 1236527 1188644 1275005 1233392 43265.76879
2 1 C-2 1094773 1284966 1424882 1268207 165691.3879
1 0.5 C-3 1413420 920830 900279 1078176 290511.3129
0.5 0.25 C-4 986848 1150559 1056754 1064720 82145.72253
0.25 0.125 C-5 887658 899227 936575 907820 25565.56197
0 0 C-6 872434 1119336 93454 695074.7 535444.4129



Calibration curves

Image:SuperTrooper.jpg

Image:Honeyhoney.jpg


Images of Our PCR Negative and Positive Controls

Image:SuperNatural.jpg

Positive

Image:KiRbY.jpg

Negative


PCR Results: PCR concentrations solved

PCR Product TUBE Label MEAN (of RAWINTDEN DROP - BACKGROUND) PCR Product Concentration (μL/mL) (Step 5 calculation) Total Dilution Initial PCR Product Concentration (μL/mL) (Step 6 Calculations)
G4 + 15179.6667 -3.27514 12 -39.30169
G4 - 18965 -3.25971 12 -39.11656
G4 1-1 3783.33333 -3.32159 12 -39.85905
G4 1-2 -18287 -3.41154 12 -40.93845
G4 1-3 44859.3333 -3.15417 12 -37.85014
G4 2-1 48412 -3.13970 12 -37.67639
G4 2-2 65845 -3.06865 12 -36.82379
G4 2-3 67970.3333 -3.05999 12 -36.71985


PCR Results: Summary

  • Our positive control PCR result was -39.85904965, -40.9384463, and -37.8501404 μg/mL.
  • Our negative control PCR result was -37.67638968, -36.82379169, and -36.71984774 μg/mL.


Observed results

  • Patient 12188 : Quantitatively, the values for the initial PCR reaction concentration were very close to both the negative and positive control values at at approximately -39 micrograms per milliliter. Qualitatively, the patient samples looked identical to the negative control; the pictures were very dark except for the poles of the drops and the beam of light.
  • Patient 54737 : Quantitatively, the initial PCR reaction concentration values were close to the negative and positive control amounts at about -36 micrograms per milliliter. Qualitatively, the patient samples looked exactly like the negative control; additionally, the photos were very dark except at the poles of the drops and the beams of light.


Conclusions

  • Patient 12188 : For samples one and two, the resulting values of the initial PCR product concentration were closer to the value of the positive control initial PCR product concentration. The third sample was closer to the negative control initial PCR concentration than to the positive control value. Therefore, since two out of the three samples were closer to the positive control, patient 12188 was positive.
  • Patient 54737 : For all three samples, the resulting values of the initial PCR product concentration were closer to the value of the negative control than to the positive control of the initial PCR product concentration. Therefore, since all three samples were closer to the negative control, patient 54737 was negative.



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