BME100 s2018:Group8 W0800 L5

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OUR TEAM

Name: Jill Leaver
Name: Hector Figueroa
Name: Auhood Alghareed


LAB 5 WRITE-UP

PCR Reaction Report

The pre-lab reading was an extremely helpful guiding tool throughout this lab. It was used for reference multiple times during the procedure. Our group did understand the difference between the first and second stop on the pipettor. The first stop is used to collect the sample, and the second stop is to dispense it. The second stop adds a puff of air when expelling the sample in order to ensure it gets all of it out. After the reaction, the eight tubes did appear to have the same amount of liquid due to the measuring precision of the micropipettor. However, after the reaction was completed and the solutions were transferred, there was still a small amount of liquid left, which we just collected by doing another round of pipetting to ensure we got all of the sample. We did not change our labeling scheme, such that it was kept consistent throughout the lab.

Fluorimeter Procedure

Imaging set-up

  1. Set up the smart phone; a Huawei 8 was used in this lab
  2. Adjust the camera settings to:

-Inactivate the flash

-Set ISO to 800 or higher

-Set white balance to auto

-Set exposure and saturation to the highest setting

-Set contrast to the lowest setting

  1. Place the smart phone onto the phone stand
  2. Prop the fluorimeter to an appropriate height
  3. After placing the drops on the slide, set a timer, close the lid of the fluorimeter, close the flap, and then take the pictures


Placing Samples onto the Fluorimeter

  1. Place the slide with the smooth side down and the rough hydrophobic side up on the fluorimeter
  2. Turn on the fluorimeter
  3. Set the camera timer for 3 seconds
  4. Place the smartphone on cradle with the camera app on
  5. Adjust the height of the fluorimeter to get a camera view of the slide
  6. Micropipette 80 µL of the SYBR Green 1 solution in between the middle circles on the first 2 clear circles in the middle of the slide
  7. Micropipette 80 µL of the sample/calibration solution onto the drop of the SYBR Green 1 Solution
  8. Adjust the slide so the light illuminates the center of the drop
  9. Adjust the distance between the smartphone and the fluorimeter
  10. Cover the fluorimeter with the light box
  11. Check to make sure the drop is focused
  12. Hit the timer button on the camera and lower the flap before the picture is taken
  13. Remove the solution from the slide and discard it into a waste container
  14. Move the slide to the next position, which is the center of the next two circles
  15. Repeat this procedure for all of the samples


Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA

5 μg/mL Sample of Calf Thymus
0.5 μg/mL Sample of Calf Thymus
0 μg/mL Sample of Calf Thymus

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-Back: Image 1 Rawintden Drop-Back: Image 2 Rawintden Drop-Back: Image 3 MEAN Standard Deviation
5 2.5 C-1 28550161 28246716 41197239 32664705.3 7390948.369
2 1 C-2 5173414 4248857 5024846 4815705.67 496493.8779
1 0.5 C-3 10347231 6550329 6415733 7771097.67 2232011.701
0.5 0.25 C-4 8922298 6630488 6636535 7396440.33 1321434.961
0.25 0.125 C-5 4597694 12864222 14610157 10690691 5348413.932
0 0 C-6 4601661 5141363 437483 3393502.33 2574171.184


Calibration curves

Negative Control Positive Control


Images of Our PCR Negative and Positive Controls, respectively.

Negative Control Positive Control


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)"
Positive 13358929 1.56 12 16.98
Negative 2956032 -1.65 12 -21.76
Patient 1-1 6110412.7 -0.63 12 -7.56
Patient 1-2 529585431 -0.7 12 -11.45
Patient 1-3 6290890.7 -0.64 12 -6.34
Patient 2-1 187006989 3.78 12 43.67
Patient 2-2 141222345 2.45 12 23.78
Patient 2-3 158333673 2.59 12 32.54


PCR Results: Summary

  • Our positive control PCR result was 16.98 μg/mL
  • Our negative control PCR result was -21.76 μg/mL


Observed results

  • Patient 58297 : This patient had bright images, indicating that there was an extensive reaction between the DNA and the SYBER green. These images resembled those of the positive control. This makes sense since our calculated values were 43.67μg/mL, 23.78μg/mL, and 32.54μg/mL, which are values close to that of the positive sample.
  • Patient 86632: This patient had dark images, indicating that the reaction between the DNA and the SYBER green was minimal. These images resembled those of the negative control. This makes sense since our calculated values were -7.56μg/mL, -11.45μg/mL, and -6.34μg/mL, which are values close to that of the negative sample.


Conclusions

  • Patient 58297 : This patient was positive. This conclusion was reached because all three of the patients samples were calculated to have DNA concentrations in the positive range. These numbers were large, indicating that the DNA concentrations in the PCR samples were high, which means that DNA was replicated in large amounts in the PCR reaction. Moreover, these samples resembled the positive control, indicating that the disease primers were able to successfully bind to the disease positive patient.
  • Patient 86632 : This patient was negative. This conclusion was reached because all three of the patients samples were calculated to have DNA concentrations in the negative range. These numbers were low, indicating that the DNA concentrations in the PCR samples were low, which means that the DNA replication did not occur. This sample resembled the negative sample, and since both samples did not light up, they are both negative.



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