OUR TEAM
LAB 5 WRITE-UP
PCR Reaction Report
Our experience pipetting the samples to set up the reaction went very well. This was likely because of the information gained from the pre-lab reading. The information regarding pipette handling techniques was especially helpful. The first stop on the pipette is for inserting liquid into the device and the second stop is to eject the liquid. The final reaction tubes appeared to have the same amount of liquid in them. There was a minimal amount of liquid left in the tubes that the DNA samples and PCR reaction mix came from. We did not have to change our labeling scheme. We made it consistent across the sections of the lab in order to keep our tubes organized.
Fluorimeter Procedure
Imaging set-up
- Adjust smartphone camera settings, a Samsung Galaxy 8 was used in this lab
-Turn off the camera flash
-Set the exposure and saturation to the highest level
-Set ISO to 800+
-Set contrast to the lowest setting
- Set the smartphone into the provided holder
- Raise the platform to the appropriate height using the materials provided
- Measure the distance between the platform and the smartphone, this should be no larger than 4 cm.
- The use of a timer for the camera is recommended, but not necessary
Placing Samples onto the Fluorimeter
- Place the slide rough-side-up in the fluorimeter
- Turn on the fluorimeter
- Place a 80 µL volume of SYBR Green 1 solution in-between the circles in the first two rows
- Place a 80 µL volume of calibration/sample solution onto the drop of SYBR Green 1 solution
- Adjust the position of the slide so that the light from the fluorimeter illuminates the drop
- Place the smartphone set up in closest possible configuration to the drop
- Take pictures
- Remove and clean slide
- Dispose of liquid in proper waste receptacle
- Place next drop in the center of the next two circles
- Repeat this procedure for each sample
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
High Calf Thymus DNA
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Low Calf Thymus DNA
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Zero Calf Thymus DNA
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Calibrator Mean Values
| Final DNA concentration in SYBR Green I solution (µg/mL) |
Sample Number |
RAWINTDEN DROP - BACKGROUND |
|
|
MEAN |
Standard Deviation
|
|
|
Image 1 |
Image 2 |
Image 3 |
|
|
| 2.5 |
C-1 |
16378810 |
12786081 |
13413884 |
14192925 |
1918880.94
|
| 1 |
C-2 |
12044159 |
12544937 |
13133152 |
12574082.67 |
545081.22
|
| 0.5 |
C-3 |
12773960 |
12931855 |
13314267 |
13006694 |
277819.31
|
| 0.25 |
C-4 |
12170806 |
11422459 |
9386428 |
10993231 |
1440960.69
|
| 0.125 |
C-5 |
4597694 |
12864222 |
14610157 |
10690691 |
5348413.93
|
| 0 |
C-6 |
4601661 |
5141363 |
4374863 |
4705962.333 |
393750.73
|
Calibration curves
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Images of Our PCR Negative and Positive Controls
Negative Control
Positive Control
PCR Results: PCR concentrations solved
| PCR Product TUBE LABEL |
MEAN (of RAWINTDEN DROP - BACKGROUND) |
PCR Product Concentration (µg /mL) (Step 5 calculation) |
Total Dilution |
Initial PCR Product Concentration (µg /mL) (Step 6 calculation)
|
| Positive |
12338929.00 |
1.45 |
12.00 |
17.36
|
| Negative |
2956034.00 |
-1.68 |
12.00 |
-20.18
|
| Patient 1-1 |
6110412.70 |
-0.63 |
12.00 |
-7.56
|
| Patient 1-2 |
5295854.30 |
-0.90 |
12.00 |
-10.82
|
| Patient 1-3 |
6290890.70 |
-0.57 |
12.00 |
-6.84
|
| Patient 2-1 |
19006989.00 |
3.67 |
12.00 |
44.03
|
| Patient 2-2 |
14122256.00 |
2.04 |
12.00 |
24.49
|
| Patient 2-3 |
15833365.00 |
2.61 |
12.00 |
31.33
|
PCR Results: Summary
- Our positive control PCR result was 17.36 μg/mL
- Our negative control PCR result was -20.18 μg/mL
Observed results
- Patient 94130 : For Patient 1 (94130), the images were all very dark both in color and in ImageJ. It was clear that the reaction between SYBER green and the DNA was very minimal because the light passing through the droplet did little to change the appearance of the droplet. In this way, the images for this patient looked similar to that of the negative control. This makes sense because the calculated concentrations were -7.56, -10.82, and -6.84 micrograms/milliliter for the three samples of Patient 1 DNA. These numbers line up closer to that of the negative sample
- Patient 76365 : For Patient 2 (76365), the images were much brighter both in ImageJ and in color. The ImageJ images for Patient 2 samples were so bright they appeared nearly white in some parts, and there was an extremely distinct green glow seen in the color images. Hence, it is clear that the reaction between SYBER green and the DNA occurred on a large scale because the light passing through the droplet excited most of the SYBER green molecules. In this way, the images for this patient looked similar to that of the positive control. This makes sense because the calculated concentrations were 44.03, 24,49, and 31.33 for the three Patient 2 samples. These numbers line up more closely with the positive control.
Conclusions
- Patient 94130 : This patient is negative. We can determine this because all 3 of the patient's samples were calculated to have DNA concentrations in the negative numbers. This means that the DNA concentrations of these PCR samples were extremely low, which in turn means that DNA replication did not occur in these samples. As shown by the positive and negative controls, if DNA replication had occurred and the sample had lit up, the patient would have been positive. However, because the calculated concentration and appearance of the sample were similar to that of the negative control, it can be determined that Patient 94130 tested negative for the disease.
- Patient 76365 : This patient is positive. We can determine this because all 3 of the patient's samples were calculated to have DNA concentrations in the higher positive range. This means that the DNA concentrations of these PCR samples were relatively high, which in turn means that DNA replication occurred on a large scale during the PCR reaction. As shown by the positive control, large-scale DNA replication means that the PCR reaction was successful and the disease positive primers were able to bind to disease positive patient DNA. Because the appearance and calculated concentration of the sample was similar to that of the positive control, it can be concluded that patient 76365 tested positive for the disease.
|
BME 100 Spring 2018
