OUR TEAM
LAB 5 WRITE-UP
PCR Reaction Report
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
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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
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Images of Our PCR Negative and Positive Controls
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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
