BME100 f2015:Group17 1030amL5

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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
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OUR TEAM

Andrew Nelson: student
Role(s)
Name: Taylor Underwood
Eric Slovak: student
Role(s)
Name: Augustas Laurin
Name: Julianna Acero
Name: student


LAB 5 WRITE-UP

PCR Reaction Report

The pre-lab was critical in helping our group perform the lab. The pipetting technique and sequence of experiments was crucial to a successful lab and the pre-lab certainly helped. We learned in this lab how to use a micro pipette. The first stop on the pipet is what is used to draw a solution in. Pushing down to the first stop and releasing in when inside of a solution allows the pipet to take in the desired amount that was previously set. Pressing down to the second stop releases all of the solution out of the pipet, it essentially clears out the pipet. The pipette tip is then disposed of and the procedure is repeated. The liquid would be attracted to the hydrophilic dots and rejected by the hydrophobic areas therefore making a nice large droplet big enough to shine the light through. The final reactions therefore had the approximately the same amount of liquid as the pipette procedure was followed. There was very little liquid left in some of the tubes. Our labeling scheme worked as we transferred the eight PCR labels exactly the same order that appeared on the original tubes to the buffer solution.

Fluorimeter Procedure

Smart Phone Camera Settings

  • Type of Smartphone: iPhone 6
    • Flash: Off
    • ISO setting: Standard iPhone Settings
    • White Balance: Standard iPhone Settings
    • Exposure: Standard iPhone Settings
    • Saturation: Standard iPhone Settings
    • Contrast: Standard iPhone Settings


Camera set-up
Place the Fluorimeter inside the black box and place the phone in a plastic cradle 7.5 cm away from the Fluorimeter. Make sure the phone camera is level with the Fluorimeter, and the camera settings are the same as listed above. Make sure camera is inside the light box for better picture quality.

  • Distance between the smart phone cradle and drop = 7.5 cm


Placing Samples onto the Fluorimeter

  1. Place a 160 microliter drop of water onto the slide in the middle of the first two rows.
  2. Place a 80 microliter drop of SYBR GREEN in the middle of the first two rows
  3. Add a 80 microliter drop of calf thymus (or water blank) to the 80 microliter drop of SYBR GREEN
  4. Use a pipettor to remove the 160 microliter drop and then repeat the previous step with varying concentrations of calf thymus


Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA

High: High.png Low: Low.png Zero: Zero.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: Image 1 RAWINTDEN DROP - BACKGROUND: Image 2 RAWINTDEN DROP - BACKGROUND: Image 3 Mean Standard Deviation
5 2.5 C-1 5767154 6962665 5890219 6206679 657588.0129
2 1 C-2 6485286 7838282 5138207 6487258 1350038.581
1 0.5 C-3 4384525 4640595 4059280 4361466 237880.2883
0.5 0.25 C-4 4630644 3785262 4511677 4309194 457621.1361
0.25 0.125 C-5 4633765 3808092 4625352 4355736 474292.559
0 0 C-6 2083069 2565366 1851459 2166631 364215.3193


Calibration curves
DotPlotCal.png Dot Plot 1: y=623539.581x+3738499.167 - Dot Plot 2: y=448922.098x+240118.003

Images of Our PCR Negative and Positive Controls

Positive: Positive1.png Negative: Negative(1).png


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)
G17 + 8004911 17.29652 12 207.55831
G17 - 4056540.33333 8.50130 12 102.01562
G17 1-1 8733667 18.91987 12 227.03847
G17 1-2 10031545 21.81097 12 261.73166
G17 1-3 10062024.33 21.87887 12 262.54639
G17 2-1 3595719.333 7.47480 12 89.69756
G17 2-2 3676232.333 7.65414 12 91.84973
G17 2-3 3749925.333 7.81830 12 93.81960


PCR Results: Summary

  • Our positive control PCR result was 207.5583 μg/mL
  • Our negative control PCR result was 102.0156 μg/mL


Observed results

  • Patient 52130 : Qualitative- the drops were bright green - Quantitative- 227.04, 261.73, 262.55 (μg/mL)
  • Patient 83165 :Qualitative- the drops were clear with no green - Quantitative- 89.70, 91.85, 93.82 (μg/mL)


Conclusions

  • Patient 52130 : This patient's results came back as 227.04, 261.73, 262.55 (μg/mL), which would indicate that this patient is positive, due to the that fact that the results are closer to the positive value of 207.5583 μg/mL
  • Patient 83165 : This patient's results came back as 89.70, 91.85, 93.82 (μg/mL), which would indicate that this patient is negative, due to the fact that the results are closer to the negative value of 102.0156 μg/mL