BME100 f2015:Group6 1030amL5

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Contents

OUR TEAM

Caroline Dolt(s)
Caroline Dolt(s)
Kamrie Ickes(s)
Kamrie Ickes(s)
Rex Moore
Rex Moore
Connor Beck
Connor Beck
Name: Chricitan B. Adane
Name: Chricitan B. Adane
Andres X. Gandara
Andres X. Gandara


LAB 5 WRITE-UP

PCR Reaction Report

Our team worked sufficiently to set up the reaction using pipetting techniques. We had 8 tubes, one being the positive control and another being the negative control. The other six tubes contained the 100 nanoliter PCR reactions. After each reaction was set up and the lids on each tube were closed tightly, we transferred them to the PCR machine. The pre-lab was beneficial because it prepared us for the use of a pipettor by listing each each step required to get successful measurements. We understood that the first stop of the pipettor should be used for obtaining the volume of solution noted on the side of the device, while the second stop is used for injecting the solution into the tubes. It is because of the accuracy of the pipettor the the final reactions had the same amount of liquid. We did not see any excess liquid in the tubes after transferring it. Labeling was also successful, as we followed the instructions given to us.

Fluorimeter Procedure

Smart Phone Camera Settings

  • Type of Smartphone: iPhone 5
    • Flash: Off
    • ISO setting: No setup option
    • White Balance: No setup option
    • Exposure: No setup option
    • Saturation: No setup option
    • Contrast: No setup option


Camera set-up
The phone was placed in the cradle and oriented to face the fluorimeter. The camera on the phone was higher than the fluorimeter so two small tupperware tops and a notebook were placed under the fluorimeter to make the camera level with the fluorimeter. A usb drive was also placed in the cradle along with the phone so the phone would not be at an angle when taking pictures of the drop. The usb drive was between the back of the phone and the cradle so the drop on the fluorimeter and the camera were on the same horizontal plane.

  • Distance between the smart phone cradle and drop = 4 cm


Placing Samples onto the Fluorimeter

  1. A slide was positioned on the fluorimeter
  2. 80 microliters of Sybr Green were added between two of the rows on the slide
  3. 80 microliters of the DNA sample were added
  4. A three second timer was set on the phone and a picture was taken
  5. Two more pictures were taken for each DNA sample
  6. The 160 microliters of Sybr Green and DNA sample were removed from the test tube using the micropipette
  7. The slide was moved so the next two rows were in line with the light

Steps 2-7 were repeated until all DNA samples had been photographed three times


Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA

(1) 5 μg/mL sample

Description of image

(2) 0.5 μg/mL sample

Description of image

(3) zero DNA

Description of image

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 RAWINTDEN DROP - BACKGROUND RAWINTDEN DROP - BACKGROUND MEAN Standard Deviation
Image 1Image 2Image 3
52.5C-17695436877881086483778374207.66666667591440.126702892
21C-29976618101392711021787510111254.6666667123044.387163061
10.5C-35960300597613963151996083879.33333333200485.185538317
0.50.25C-45413972586586251917205490518343527.766895196
0.250.125C-54962662535143352238475179314198174.452634541
00C-627672433014607828221954890.666666671625969.88184294

Calibration curves


Images of Our PCR Negative and Positive Controls

(1) G6- Control

Description of image

(2) G6+ Control

Description of image


PCR Results: PCR concentrations solved

PCR Product TUBE LABEL MEAN (of RAWINTDEN DROP _ BACKGROUND PCR Product Concentration (µg /mL)

(Step 5 calculation)

"Total Dilution "Initial PCR Product Concentration

(µg /mL) (Step 6 calculation)

(Step 6 calculation)"
G6+65882651.5882651219.05918
G6-5219016.66670.21901667122.6282
G6 1-166230921.6230921219.477104
G6 1-23326347.3331.6736526671220.083832004
G6 1-33174961.3331.8250386671221.900464004
G6 2-193162794.3162791251.795348
G6 2-291774474.1774471250.129364
G6 2-38846335.66673.84633566671246.1560280004


PCR Results: Summary

  • Our positive control PCR result was 19.05918 μg/mL
  • Our negative control PCR result was 2.6282 μg/mL


Observed results

  • Patient 41418 : The photos for this patient show green drops. The average concentration was 20.48713334 μg/mL.
  • Patient 39567: The photos for this patient show clear drops. The average concentration was 49.36024667 μg/mL.


Conclusions

  • Patient 41418 : This patient matches the positive photo. The average of this patient is very close to the positive control, supporting that the SNP is present in this patient's DNA.
  • Patient 39567 : This patient matches the negative photo. However, the concentration of this patient is closer to the positive control concentration than the negative control concentration. Visually, the patient is negative but quantitatively, the patient is positive. These results are inconclusive and a retest would be advised.



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