BME100 f2017:Group16 W1030 L5

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OUR TEAM

Name: Alfred Abraham
Name: Alexia Kamau
Name: Hannah Corea
Name: Jaynee Monarrez
Name: Michelle Mungaray


LAB 5 WRITE-UP

LAB C: PCR Reaction Report

Our teams experience with pipetting the samples to set up the reaction was very good. There were few mistakes made which made the lab run smoothly overall. The pre-lab reading did help us figure out how to set up this part of the lab and acted as a guide for us as we made our samples. We did have one person in our group who had experience with this type of pipetting technique so her input was very helpful and she was able to show us how to do this. After looking over the pre-lab reading, we did understand the difference between the first and second stop on the pipettor. After pipetting these samples, the final reactions did end up having the same amount of liquid in them, which provided for more accurate results. As well as this, there was no liquid left in the tubes that the DNA samples and PCR reaction mix were in. The labeling process was done before the pipetting began because it would have been difficult to label when there was liquid in each small tube. This part was more challenging because the tubes were so small, however we managed to label each tube with a small number on the top. Overall, this section of the lab did not present too much difficulty and it was an important process that led into the continuation of this experiment.

Lab D: Fluorimeter Procedure

Imaging set-up
First, the proper materials were gathered: a fluorimeter, a micropipette, micropipette tip boxes, 3 glass slides, and a smartphone (iPhone 7 plus). In order to setup the fluorimeter device, one of the three glass slides was placed on top of the device with the smooth side facing down and the rough side facing up. The smartphone was then placed onto a phone stand about 11 cm away from the fluorimeter on top of 2 tip boxes in order to level the camera with the top of the device.


Placing Samples onto the Fluorimeter

  1. Turn the fluorimeter device on.
  2. Place one of the glass slides in the fluorimeter with the smooth side facing down and the rough side facing up.
  3. Adjust the height of the phone stand by placing it on top of tip boxes in order to get the camera in level with the fluorimeter.
  4. Using a micropipette, place 80 ul of SYBR Green I along the middle column of dots, the drop should be 2 circles wide.
  5. Then add 80 ul of the calibration solution (0, 0.25, 0.5, 1.0, 2.0, 5.0) to the drop of SYBR Green I on the glass slide.
  6. If needed, move the slide so that the drop is illuminated by the fluorimeter light at its center.
  7. Move the phone stand closer if needed to get a clear and close picture of the drop, record the distance. Take 3 pictures for each calibration sample.


Lab D:Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA :


5 μg/mL sample
0.5 μg/mL sample
Zero DNA


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 OF DROP- RAWINTDEN OF BACKGROUND: Image 1 RAWINTDEN OF DROP- RAWINTDEN OF BACKGROUND: Image 2 RAWINTDEN OF DROP- RAWINTDEN OF BACKGROUND: Image 3 Mean Standard Deviation
0 0 C-1(0) 4882447 483355 4304333 3223378.33 2390470.634
0.25 0.125 C-2(0.25) 5417794 4557788 4444092 4806558 532389.6889
0.5 0.25 C-3(0.5) 5130128 4829759 5290832 5083573 234035.4756
1 0.5 C-4(1.0) 6099350 6024540 5408529 5844139.667 379099.7543
2 1 C-5(2.0) 5911222 7074151 7306379 6763917.333 747528.9022
5 2.5 C-6(5.0) 8115702 7440784 6109844 7222110 1020651.908


Calibration curves


Calibration Curve 1(1).png Calibration Curve 2(1).png

Images of Our PCR Negative and Positive Controls :

Positive Control
Negative Control


PCR Results: PCR concentrations solved

Screenshots (15).png


PCR Results: Summary

  • Our positive control PCR result was __30.72__ μg/mL
  • Our negative control PCR result was __32.76__ μg/mL


Observed results

  • Patient 35640: image did not display a fluorescent green color within the droplet despite the concentration being altered between 0 μg/mL,0.25 μg/mL 0.5μg/mL , 1.0 μg/mL, 2.0μg/mL and lastly 5.0 μg/mL.
  • Patient 13601 : also did not display a green hue


Conclusions

  • Patient 13601 : was negative because compared to the positive and negative control's the drops did not turn green when mixed with SYBR green.
  • Patient 35640 : was negative as well because the drops did not turn green when mixed with SYBR green.