Since everyone in the group watched the tutorials or have done previously work with pipettes, the pipettes were not a problem to work with. Some of the group members needed a little review for the difference between the first and second stop for the pipettes. The first stop was pressed before having any liquid touching the pipette, which afterward, the pipette would be released, which would bring the liquid in to the pipette. The liquid was not the same in each of the final reactions, which made there be a slight leftover liquid. Since our group was actually organized, the labeling scheme that we used worked well.
Fluorimeter Procedure
Web camera set-up
Type of Smartphone:iPhone 6s
Flash:No Flash
ISO setting: 800
White Balance: No
Exposure: No
Saturation: No
Contrast: No
Placing Samples onto the Fluorimeter
The calibration method used to begin this experiment is placing 160 μL drop of water in the middle of the first rows located on the slide using a pipettor
The excitation light got the Blue LED light was turned on using the switch
The camera on the smartphone being used was prepared and placed in the cradle at a right angle of the slide. Necessary adjustments were made in order to capture the best angle of the drop
The cradle containing the phone was then adjusted so that the distance between it and the first two rows on the slide were as close as possible without the image becoming blurry or disturbed.
The distance between the smartphone cradle and the drop were recording using the ruler provided, with lots of caution.
An 80 μL drop of SYBR GREEN I was placed in the middle of the first two rows on the slide. Then 80 μL of one of the calf thymus (water blank) solutions were added. After this occurred, the sample on the slide was now considered a drop.
The light box provided was placed over the fluorimeter and the camera, removing as much stray light as possible.
Three images were taken using the smartphone, being careful and checking that the drop was focused in every image.
Remove the light box as careful as possible without moving the smart phone.
Remove the 160 μL drop from the surface using the pipettor and move the slide to the next position.
Repeat steps 5-10 using different concentrations of calf thymus DNA
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
Order of images left to right: 5 μg/mL, .5 μg/mL, and 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 DROP - BACKGROUND
MEAN
Standard Deviation
Image 1
Image 2
Image 3
5
2.5
C-1
784214
810886
820724
805274.6667
18890.74698
2
1
C-2
720784
754188
765290
746754
23165.58387
1
0.5
C-3
720913
747526
753916
740785
17503.72283
0.5
0.25
C-4
721951
698932
686966
702616.3333
17781.12174
0.25
0.125
C-5
642089
626108
663498
643898.3333
18760.55144
0
0
C-6
728567
720941
659668
703058.6667
37770.37774
Calibration curves
Images of Our PCR Negative and Positive Controls
From left to right: Positive and Negative controls
PCR Results: PCR concentrations solved
PCR Product TUBE LABEL
MEAN (of RAWINTDEN DROP - BACKGROUND)
PCR Product Concentration
Total Dilution
Initial PCR Product Concentration
Positive Control
2665317
78.85864062
12
946.3036875
Negative Control
1746504.33
42.23066893
12
506.7680271
1-1
2670103
79.04943193
12
948.5931832
1-2
2721205
81.08658561
12
973.0390273
1-3
2531403
73.52023121
12
882.2427746
2-1
1961057.33
50.78370859
12
609.4045031
2-2
1849168.67
46.32332749
12
555.8799298
2-3
1936383.33
49.80009288
12
597.6011146
PCR Results: Summary
Our positive control PCR result was 946.3036875 μg/mL
Our negative control PCR result was 506.7680271 μg/mL
Observed results
Patient 20710 :The images of this patient a dark green light from the photos taken, which was very similar to the positive droplets. These images showed luminescence inside of them. The observed concentration of the droplets from this patient had an average of 946.304 μg/mL.
Patient 35451 :The images of this patient were dark with very little if not any luminescence. They showed very little green light compared to the positive droplets. The observed concentration for this patient had an average of 506.7680 μg/mL.
Conclusions
Patient 20710 :While looking at the data analysis and the pictures, this patient appears to be similar to the positive control due the green light and it what somewhat luminescence. The patient had an average of 921.627 μg/mL, which is very close to the positive sample of 946.304 μg/mL. This means that the patients was probably positive.
Patient 35451 :When looking at the data, this patient appears to be similar to the negative control due to the lack of green light within each droplet. The average concentration of 587.629 μg/mL was very close to the concentration of the negative sample which was 506.768 μg/mL. Therefore, this patient was negative.
BME 100 Spring 2016
