BME100 f2017:Group7 W0800 L5

From OpenWetWare
Jump to navigationJump to search
Owwnotebook icon.png BME 100 Fall 2017 Home
People
Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
Course Logistics For Instructors
Photos
Wiki Editing Help
BME494 Asu logo.png


OUR TEAM

Name: Wesley Groves
Name: Michael Zou
Name: Sayyed Ourmazd Mohseni
Name: Miguel Almanza Lopez
Name: Abdulmonem Alshammari
Name: student


LAB 5 WRITE-UP

PCR Reaction Report

The pipetting of the samples was successful because there was little to no remaining liquid in the plastic test tubes. The pre-lab readings were extremely helpful because it introduced all of the concepts from this experiment before actually participating in the investigation. The pipetting simulation gave insight to the first and second stops for the pipette; the first being for extracting the liquid and the second for expelling of the liquid. All of the final reaction vials had the same amount of liquid indicating that the contents were added accurately. The labeling scheme consists of a G7 on both separate test tube racks and the patient and sample number on each individual test tube. For example, patient 1 sample 2 would be indicated '1-2.'

Fluorimeter Procedure

Imaging set-up

First, our fluorimeter set up consisted of placing the fluorimeter on a level surface surrounded by a black box to eliminate as much light as possible. The fluorimeter was also placed on top of two test tube boxes to level the iPhone 6 camera with the drop of sample. The iPhone 6 was stabilized by a phone holder which was placed 6cm away from the fluorimeter. When the set up was complete, there was complete darkness within the black box with the exception of the blue light(~470nm wavelength) emitted by the fluorimeter.


Placing Samples onto the Fluorimeter

  1. Step one: Complete set up listed above, and insert the slide with the hydrophobic side facing away from the fluorimeter.
  2. Step two: Using a micropipette, place 80uL of SYBR green between the between the two middle dots on the hydrophobic side of the slide.
  3. Step three: Using a micropipette, place 80uL of the PCR mix of patient 1 between the between the two middle dots on the hydrophobic side of the slide, right on top of the SYBR green drop.
  4. Step four: Turn on the fluorimeter and close the black box. Let the timed phone take three pictures.
  5. Step four: Repeat steps 1-4 for the other 2 samples of patient 1, the 3 samples for patient 2, and the positive and negative controls samples.


Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA

The following images are of the high concentration (5 μg/mL), low concentration (0.5 μg/mL), and the water droplet respectively. Each image had the color channels split and only the green channel was used to gather data.

G7 High concentration.png

5 μg/mL concentration


G 7 Low concentration.png

.5 μg/mL concentration


G7 Water pic.png

Zero concentration


Calibrator Mean Values

Initial Concentration of 2X DNA Solution MEAN
0 1188119.333
0.25 6460768.333
0.5 8508822
1 9553813.667
2 13833207.33
5 20208950.67


Calibration curves

G7 Cal curve 1.png

Calibration Curve 1

G7 Cal curve 2.png

Calibration Curve 2

Images of Our PCR Negative and Positive Controls

The following images were taken of the positive and negative controls respectively. They are taken from a green color channel that was split using ImageJ.

G 7 PCR Positive control.png


G7 PCR Negative control.png


PCR Results: PCR concentrations solved


PCR Tube Label MEAN RAWINTDEN DROP-BACKGROUND PCR Product Concentration (μg/mL) Total Dilution Initial PCR Product Concentration (μg/mL)
P 12735917 1.759535019 12 21.11442022
N 2218279 -0.2100601124 12 -2.520721348
1-1 1060260 -0.426917603 12 -5.123011236
1-2 3178386 -0.03026479401 12 -0.3631775281
1-3 2365896 -0.1824164794 12 -2.188997753
2-1 1160862 -0.4080782772 12 -4.896939326
2-2 2179850 -0.2172565543 12 -2.607078652
2-3 4986314 0.3082985019 12 3.699582022



PCR Results: Summary

  • Our positive control PCR result was 21.11442022 μg/mL
  • Our negative control PCR result was -2.520721348 μg/mL

The negative concentration observed for the negative control above could be due to an error found in the calibration curves. However, the difference between the positive and negative concentrations support the validity of this experiment.


Observed results

  • Patient 97085 : No observed fluorescence the mean initial PCR DNA concentration observed was -2.5584 μg/mL
  • Patient 52014 : No observed fluorescence the mean initial PCR DNA concentration observed was -1.26815 μg/mL


Conclusions

  • Patient 97085: Negative
  • Patient 52014: Negative


Both of the patients displayed extremely low concentrations of their initial PCR product concentrations. These results support the statement that neither of the patients have the single nucleotide polymorphism that this experiment is testing for.