BME100 f2016:Group8 W8AM L5

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Contents

OUR TEAM

Name: Juan Pablo Robayo
Name: Juan Pablo Robayo
Name: Dhantin Kumar
Name: Dhantin Kumar
Name: Jasmine Davis
Name: Jasmine Davis
Name: Andre Bordeleau
Name: Andre Bordeleau
Name: Blake Sentyrz
Name: Blake Sentyrz


LAB 5 WRITE-UP

PCR Reaction Report

Pipetting the samples into the PCR reaction was not a difficult task to accomplish. The first step in pipetting is to set the volume to 250 uL. This is done by twisting the top of the pipette until it reaches this measurement. Then we attach a micro pipette tip to the pipette by pressing down on the pipette tip. Then one has to press down on the plunger on the top of the pipette until it reaches the first stop and no further, and hold the plunger down as the pipette is lowered into the sample. Then the plunger is slowly released to its start position, and as this is happening, the pipette tip sucks up the liquid. After waiting one or two seconds and making sure the tip has no air bubbles, the tip is slowly and vertically pulled out of the tube and lowered into the PCR reaction tube that will hold our samples when we put it in the PCR machine. Then the plunger is pressed down to the second stop so that all of the sample is dispelled into the PCR reaction tube, and after waiting one or two seconds to make sure all the liquid is dispelled into the tube, the tip is removed from the tube and the plunger is slowly released. After the sample is in the PCR reaction tube and the tube is closed, the pipette tip is ejected from the pipette by pressing down on the eject plunger on the side of the pipette into a disposal bag. Overall, our experience with pipetting went well as we followed the instructions and knew what we were doing. The pre-lab reading did help because it helped give us a basic understanding of how the pipette worked and how pipette technique works. The pipette videos also helped us understand the difference between the first and second stop on the pipettor, but we also had previous knowledge on how the pipettor worked and proper pipetting technique. The final reactions did have the same amount of liquid because the precise amount of 80 uL of the sample and the PCR reaction mixtures were added into the reaction tube with the pipettor. However, there could've been errors made, as some sample pipetting could have had some air bubbles in the tips causing some inaccurate amount of liquid. There was no concentrated sample solution left in the sample tubes, but there was some diluted sample patient solution left in the tubes that were pipetted and some liquid left in the tubes that held the PCR reaction mix. We didn't change any of the labeling scheme, as all the tubes were properly labeled with the patient samples, positive control, and negative control.

Fluorimeter Procedure

Imaging set-up

1) Acquire a smartphone

2) Open camera app, set the timer to three seconds, and turn the camera flash off if possible.

3) Place smartphone in cradle, and adjust height if needed.

4) Camera set up: vertically align camera in cradle so that camera lens is at the same level as where the drop will be placed.



Placing Samples onto the Fluorimeter

  1. Place fluorimeter on table and then turn it on. With the smooth side facing down, put the slide into the fluorimeter.
  2. After setting up the camera, put 80 uL of the Syber Green 1 solution onto the middle of the slide in between the two clear dots.
  3. Then on top of the Syber Green 1 solution, place 80 uL of the sample or the calibration.
  4. Adjust smartphone distance and light placement so that the camera is able to focus on the drop and that the light is illuminating the middle of the drop.
  5. Place box over the fluorimeter, but keep the flap open to check and make sure that the camera on the smartphone is focused on the drop.
  6. After checking the focus, press the timer and close the box.
  7. Once the picture has been taken, remove the 160 uL solution from the slide and put it in the liquid waste container. Move the slide over to the next two circles, and repeat.
  8. Once finished with the slides, place them in the SHARPS container.


Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA

Image Sample
Drop: Zero DNA
Drop: Zero DNA
0 µg/mL
Drop: 0.5 µg/mL
Drop: 0.5 µg/mL
0.5 µg/mL
Drop: 5 µg/mL
Drop: 5 µg/mL
5 µg/mL



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 470452 477925 636999 528458.6667 94072.92066
2 1 C-2 366195 231670 574528 390797.6667 172747.9969
1 0.5 C-3 668759 798695 243652 570368.6667 290307.899
0.5 0.25 C-4 218300 360857 623870 401009 205744.7291
0.25 0.125 C-5 233011 293291 576480 367594 183394.2211
0 0 C-6 311109 301772 320085 310988.6667 9157.093007


Calibration curves

Image:calibration1.png

Image:calibration2.png


Images of Our PCR Negative and Positive Controls

Image Sample
Negative Control
Positive Control


PCR Results: PCR concentrations solved

PCR PRODUCT TUBE LABELS MEAN (OF RAWTINDEN DROP - BACKGROUND) PCR PRODUCT CONCENTRATION (µL/mL) (STEP 5 CALCULATION) TOTAL DILUTION INITIAL PCR PRODUCT CONCNETRATION
G# + 812425.67 9.63144335 3 28.8943301
G# - 267475.33 0 3 0
G# 1-1 243416.67 0 3 0
G#1-2 384795.33 0.23689734 3 0.71069202
G#1-3 646642.33 5.98937433 3 17.968123
G#2-1 262985.67 0 3 0
G#2-2 321939.67 0 3 0
G#2-3 605800.67 5.0921301 3 15.2763903

PCR Results: Summary

  • Our positive control PCR result was 5.000 μg/mL
  • Our negative control PCR result was 0.250 μg/mL


Observed results

  • Patient 45539 : The reaction came out positive and looked blue and with a major tint of fluorescent green after the solutions of 5 ug/ml and 2 ug/ml were added.
  • Patient 33494 : Reaction came out negative and looked almost blue entirely blue and with a slight tint in fluorescent green mixed with the blue. This was done using the solution of 0.250 ug/ml and a blank solution.


Conclusions

  • Patient 45539 : The patient's results and the value for the positive control were the same; they are both 5.0 μg/mL Because the patient's results were equal to that of the positive control, it can be inferred that the patient is also positive and therefore has the disease.
  • Patient 33494 : The patient's results and the value for the negative control were the same; they were both 0.25 μg/mL. Because the patient's results were equal to that of the negative control, it can be inferred that the patient is also negative and therefore does not have the disease.



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