OUR TEAM
LAB 5 WRITE-UP
PCR Reaction Report
The pre-lab was critical in helping our group perform the lab. The pipetting technique and sequence of experiments was crucial to a successful lab and the pre-lab certainly helped. We learned in this lab how to use a micro pipette. The first stop on the pipet is what is used to draw a solution in. Pushing down to the first stop and releasing in when inside of a solution allows the pipet to take in the desired amount that was previously set. Pressing down to the second stop releases all of the solution out of the pipet, it essentially clears out the pipet. The pipette tip is then disposed of and the procedure is repeated. The liquid would be attracted to the hydrophilic dots and rejected by the hydrophobic areas therefore making a nice large droplet big enough to shine the light through. The final reactions therefore had the approximately the same amount of liquid as the pipette procedure was followed. There was very little liquid left in some of the tubes. Our labeling scheme worked as we transferred the eight PCR labels exactly the same order that appeared on the original tubes to the buffer solution.
Fluorimeter Procedure
Smart Phone Camera Settings
- Type of Smartphone: iPhone 6
- Flash: Off
- ISO setting: Standard iPhone Settings
- White Balance: Standard iPhone Settings
- Exposure: Standard iPhone Settings
- Saturation: Standard iPhone Settings
- Contrast: Standard iPhone Settings
Camera set-up
Place the Fluorimeter inside the black box and place the phone in a plastic cradle 7.5 cm away from the Fluorimeter. Make sure the phone camera is level with the Fluorimeter, and the camera settings are the same as listed above. Make sure camera is inside the light box for better picture quality.
- Distance between the smart phone cradle and drop = 7.5 cm
Placing Samples onto the Fluorimeter
- Place a 160 microliter drop of water onto the slide in the middle of the first two rows.
- Place a 80 microliter drop of SYBR GREEN in the middle of the first two rows
- Add a 80 microliter drop of calf thymus (or water blank) to the 80 microliter drop of SYBR GREEN
- Use a pipettor to remove the 160 microliter drop and then repeat the previous step with varying concentrations of calf thymus
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
High:
Low:
Zero:
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: Image 1 |
RAWINTDEN DROP - BACKGROUND: Image 2 |
RAWINTDEN DROP - BACKGROUND: Image 3 |
Mean |
Standard Deviation
|
| 5 |
2.5 |
C-1 |
5767154 |
6962665 |
5890219 |
6206679 |
657588.0129
|
| 2 |
1 |
C-2 |
6485286 |
7838282 |
5138207 |
6487258 |
1350038.581
|
| 1 |
0.5 |
C-3 |
4384525 |
4640595 |
4059280 |
4361466 |
237880.2883
|
| 0.5 |
0.25 |
C-4 |
4630644 |
3785262 |
4511677 |
4309194 |
457621.1361
|
| 0.25 |
0.125 |
C-5 |
4633765 |
3808092 |
4625352 |
4355736 |
474292.559
|
| 0 |
0 |
C-6 |
2083069 |
2565366 |
1851459 |
2166631 |
364215.3193
|
Calibration curves
 Dot Plot 1: y=623539.581x+3738499.167 - Dot Plot 2: y=448922.098x+240118.003
Images of Our PCR Negative and Positive Controls
Positive:  Negative: 
PCR Results: PCR concentrations solved
| PCR Product TUBE Label |
MEAN (of RAWINTDEN DROP - BACKGROUND) |
PCR Product Concentration (μL/mL) (Step 5 calculation) |
Total Dilution |
Initial PCR Product Concentration (μL/mL) (Step 6 Calculations)
|
| G17 + |
8004911 |
17.29652 |
12 |
207.55831
|
| G17 - |
4056540.33333 |
8.50130 |
12 |
102.01562
|
| G17 1-1 |
8733667 |
18.91987 |
12 |
227.03847
|
| G17 1-2 |
10031545 |
21.81097 |
12 |
261.73166
|
| G17 1-3 |
10062024.33 |
21.87887 |
12 |
262.54639
|
| G17 2-1 |
3595719.333 |
7.47480 |
12 |
89.69756
|
| G17 2-2 |
3676232.333 |
7.65414 |
12 |
91.84973
|
| G17 2-3 |
3749925.333 |
7.81830 |
12 |
93.81960
|
PCR Results: Summary
- Our positive control PCR result was 207.5583 μg/mL
- Our negative control PCR result was 102.0156 μg/mL
Observed results
- Patient 52130 : Qualitative- the drops were bright green - Quantitative- 227.04, 261.73, 262.55 (μg/mL)
- Patient 83165 :Qualitative- the drops were clear with no green - Quantitative- 89.70, 91.85, 93.82 (μg/mL)
Conclusions
- Patient 52130 : This patient's results came back as 227.04, 261.73, 262.55 (μg/mL), which would indicate that this patient is positive, due to the that fact that the results are closer to the positive value of 207.5583 μg/mL
- Patient 83165 : This patient's results came back as 89.70, 91.85, 93.82 (μg/mL), which would indicate that this patient is negative, due to the fact that the results are closer to the negative value of 102.0156 μg/mL
|
BME 100 Fall 2015
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