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
Turn the fluorimeter device on.
Place one of the glass slides in the fluorimeter with the smooth side facing down and the rough side facing up.
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.
Using a micropipette, place 80 ul of SYBR Green I along the middle column of dots, the drop should be 2 circles wide.
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.
If needed, move the slide so that the drop is illuminated by the fluorimeter light at its center.
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
Images of Our PCR Negative and Positive Controls :
Positive Control
Negative Control
PCR Results: PCR concentrations solved
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.
BME 100 Fall 2017
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