OUR TEAM
LAB 5 WRITE-UP
PCR Reaction Report
Reaction Summary
Our team worked together to preform this experiment with the most accuracy and precision possible. Before the lab, everyone in the group completed the pre-lab material, including watching the videos and completing the virtual lab activity. The videos served very well to show us the proper technique for pipetting, from the very first step to the last. The videos began with proper handling of the materials, and continued through the attachment of the tip, the withdrawal of the liquid from the first container, and then the distribution of the liquid into the second container. The main takeaway from the videos was the distinction of the first stop and the second stop on the micropipette. If the experimenter goes to the wrong stop, the experiment can be ruined. The virtual lab served to show us the purpose of these techniques, and gave us an application to relate the new information to.
While completing our experiments, we followed proper procedure very well. We had minimal error in differentiating between the first and second stop on the micropipette, and completed the combination of the samples fairly well. It did seem that all of the tubes had the same amount of liquid when they were transferred, and it didn't seem that there was marginal error in this. However, it is important to learn from this that it would be very hard to fix an experimental error in this case. Since the liquid used in this experiment is clear, if we had forgot any liquid in the tubes after combining them, we might not notice this error unless we looked very closely. This creates some room for error further than just the misuse of the pipette itself, making the pre-lab materials and proper practice even more prominent. Thankfully we avoided error, used the same labeling scheme, and preformed a decent experiment.
Fluorimeter Procedure
Imaging set-up
We gathered all the materials needed including glass slides, lightbox, stand for the smartphone(iPhone). We adjusted our phone with the stand 4cm away from the sample which was placed on a glass slide under the lightbox with the cover to make sure that the sample did not get any light from outside. We set the timer on camera at 10 seconds to make sure we get clear pictures.
Placing Samples onto the Fluorimeter
- Fluorimeter slides were obtained (Note: handle slides at all times with gloved hands only).
- The smooth side was identified (glass coated) on first slide and then it was placed in fluorimeter with the glass side facing down.
- The camera was set-up per above, with the height adjusted as necessary to obtain edge-on view.
- Utilizing a micropipette, 80uL of SYBR Green I solution was placed on the first pair of clear circles on the slide, starting in the middle row, nearest to camera. Placement resulted in a single, cohesive drop (Note: proper pipette protocol was followed at all times, with pipette tips being changed between samples to prevent contamination).
- Utilizing a micropipette, 80uL of Sample/Calibration solution was placed on top of the SYBR Green I drop. Solution remained a single cohesive drop.
- The fluorimeter light beam from was verified to strike the center of drop.
- A 4cm distance was measured form the fluorimeter to locate proper phone placement. Phone was placed accurately.
- The lightbox was then placed over the fluorimeter and phone stand while leaving the side flap open.
- Drop was visualized through phone and a focused, edge-on view obtained.
- The timer was initiated and the flap closed.
- After the elapsed time (10sec), the flap was raised and image accuracy was verified.
- Utilizing a micropipette, the 160uL drop was removed and discarded in the liquid waste container.
- The slide was then prepared for the next sample by adjusting the slide position so that the next pair of circles in the center row were centered on the fluorimeter light beam.
- The above steps were repeated for all calibration and patient samples as directed by the workbook.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
High-5mcg Low-0.5mcg Zero-0mcg
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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
|
| 5 |
2.5 |
C-1 |
9477729 |
7819480 |
1658249 |
6318486 |
4120170.398
|
| 2 |
1 |
C-2 |
11289575 |
11220399 |
11673267 |
11394414 |
243958.4306
|
| 1 |
0.5 |
C-3 |
9970626 |
9621280 |
8986280 |
9526062 |
499033.1778
|
| 0.5 |
0.25 |
C-4 |
6867496 |
6824316 |
6531089 |
6740967 |
183037.4532
|
| 0.25 |
0.125 |
C-5 |
10119487 |
9281028 |
10035668 |
9812061 |
461793.7218
|
| 0 |
0 |
C-6 |
4532101 |
5042459 |
4723820 |
4766127 |
257795.8739
|
Calibration Curves
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Images of Our PCR Negative and Positive Controls
Negative Control Positive Control
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PCR Results: PCR concentrations solved
| PCR Product TUBE LABEL |
MEAN (of RAWINTDEN DROP - BACKGROUND) |
PCR Product Concentration (µg /mL) |
Total Dilution |
Initial PCR Product Concentration (µg /mL)
|
| G14 + |
9948075.33 |
1.29905024 |
12 |
15.58860288
|
| G14 - |
10291999 |
1.34396085 |
12 |
16.1275302
|
| G14 1-1 |
8311933.67 |
1.113033 |
12 |
13.1796672
|
| G14 1-2 |
8423390.33 |
1.113033 |
12 |
13.356396
|
| G14 1-3 |
11764224.3 |
1.53620855 |
12 |
18.4345026
|
| G14 2-1 |
11045448.7 |
1.44234862 |
12 |
17.30818344
|
| G14 2-2 |
8608675 |
1.12414723 |
12 |
13.48976676
|
| G14 2-3 |
12158640 |
1.58771256 |
12 |
19.05255072
|
PCR Results: Summary
- Our positive control PCR result was 15.6 μg/mL
- Our negative control PCR result was 16.1 μg/mL
Observed results
- Patient 62160: Within the images, the patient showed that he may have been within the spectrum of being positive, yet unfortunately our data was compromised causing the results when calculated to appear inconclusive.
- Patient 19010: Much like the previous patient, the patient's DNA when photographed showed signs of being positive, yet when the results of the information given were calculated within the graph the results showed to be inconclusive.
Conclusions
- Patient 62160: Within the results for this patient the values were rather close to the values of our positive control, however, the values were rather skewed due to the data being compromised causing the final results to be inconclusive.
- Patient 19010: This patient's values were extremely close to the positive control's values, yet the information was compromised causing the results to be inconclusive.
|
BME 100 Fall 2018
