OUR TEAM
LAB 5 WRITE-UP
PCR Reaction Report
The pre-lab report was very helpful in understanding how a micropipettor is correctly used. It was important for the group to be able to use the pipettor correctly because they are easily broken and very expensive to replace. However, some of the members of our group had previous experience with micropipettors and the pre-lab was slightly repetitive for those members. The difference between the first and second stop is critical in ensuring accurate measurements with the micropipettors. The first stop is used to collect the solution initially, then the second stop is used to add an extra puff of air when expelling the solution to get all of the solution out. If the micropipettor was pushed to the second stop before the solution is collected then the volume in the micropipettor would be higher than the set volume. After the reaction, the eight tubes did not have noticeably different amounts of liquid in the tubes. This is most likely due to the preciseness of the micropipettor. However, after the solutions were transferred, it was found that two of the tubes had a very small amount of liquid left at the bottom. It was a very small amount and most likely equivalent to around 1 ul. When labeling the new tubes, the labeling scheme was kept the same in order to keep consistency within the lab. Overall, the team had a successful time pipetting the samples in the lab.
Fluorimeter Procedure
Imaging set-up
SmartPhone 1: Samsung Galaxy s7 Model
Optional SmartPhone 2: Iphone 7 Model
Use Camera menu to first:
A. Inactivate the Flash
B. Set ISO to 800 (or higher, if possible)
C. Set white balance to auto
D. Set exposure to highest setting
E. Set saturation to the highest setting
F. Set contrast to the lowest setting
A 160 microliter drop of water was placed in the middle of the first two rows of the slide using a pipettor and generated a ball-like shape. The excitation light using the switch for Blue LED and the camera of the selected smartphone with desired settings was then turned on. Afterward, the smartphone was placed in the provided cradle on its side at a right angle. The height of the fluorimeter was adjusted using the plastic trays so the camera would take a picture of the drop sideways. The distance between the smartphone on its cradle and the first two rows of the slide were adjusted to be as close as possible without making the camera unfocused (~4 cm), and the distance was recorded.
Placing Samples onto the Fluorimeter
- Obtain a tray of sample tubes from the instructors.
- Place an 80 microliter drop of SYBR GREEN 1 in the middle of the first two rows of the slide using a micropipette.
- Place 80 uL of the sample/calibration solution on top of the SYBR GREEN 1 drop
- Align the drop by moving the provided slide so that the blue LED light is focused by the drop to the middle of the black fiber optic fitting
- Place smartphone by the fluorimeter to view the drop in focus about 4 cm away.
- Cover smartphone with lightbox, but keep one flap open
- Make sure drop is focused again before pressing timer on smartphone
- Press timer button on the camera and lower flap before taking the focused picture
- Remove the 160 uL drop from the slide and discard liquid waste in hazardous waste container
- Move the slide to the next position (center of next two circles)
- Repeat above procedures until all five possible measurement positions on the slide have been used.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
High:
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Low:
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Zero:
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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
|
|
|
|
|
Image 1 |
Image 2 |
Image 3 |
|
|
| 5
|
2.5 |
C-1 |
21921981 |
25872960 |
22867624 |
23554188.33 |
2063028.527
|
| 2
|
1 |
C-2 |
35354939 |
37461648 |
32950117 |
35255568 |
2257406.463
|
| 1
|
0.5 |
C-3 |
25936842 |
27899199 |
27930166 |
27255402.33 |
1142011.713
|
| 0.5
|
0.25 |
C-4 |
2446493 |
24552349 |
26266262 |
17755034.67 |
13285253.49
|
| 0.25
|
0.125 |
C-5 |
15757539 |
27809168 |
28260823 |
23942510 |
7091989.196
|
| 0
|
0 |
C-6 |
-967417 |
5054019 |
-817917 |
1089561.667 |
3434134.392
|
Calibration curves
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Images of Our PCR Negative and Positive Controls
Positive Control:
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Negative Control:
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PCR Results: PCR concentrations solved
| PCR Product TUBE LABEL |
RAWINTDEN DROP - BACKGROUND |
|
|
MEAN |
Standard Deviation
|
|
|
Image 1 |
Image 2 |
Image 3 |
|
|
| G3+
|
32200998 |
32223113 |
32393667 |
32272592.67 |
105434.8801
|
| G3-
|
1225862 |
19092342 |
16687908 |
12335370.67 |
9695937.955
|
| G31-1
|
23766759 |
16591241 |
20521587 |
20293195.67 |
3593207.007
|
| G31-2
|
10904910 |
10373246 |
10295179 |
10524445 |
331796.3627
|
| G31-3
|
7490520 |
6933265 |
9730834 |
8051539.667 |
1480761.756
|
| G32-1
|
17345036 |
17390382 |
18851566 |
17862328 |
857005.2103
|
| G32-2
|
15701872 |
14114444 |
20157989 |
16658101.67 |
3133191.731
|
| G32-3
|
20154600 |
12304431 |
16748398 |
16402476.33 |
3936500.309
|
| TUBE LABEL |
MEAN (RAWINTDEN DROP- BG) |
"PCR Conc. (µg /mL) |
Total Dilution |
"Initial PCR Conc. (µg /mL)"
|
| G3+
|
3227259.7 |
-0.67727403 |
12 |
-8.12728836
|
| G3-
|
12335370.7 |
0.23353707 |
12 |
2.80244484
|
| G31-1
|
20293195.7 |
1.02931957 |
12 |
12.35183484
|
| G31-2
|
10524445 |
0.0524445 |
12 |
0.629334
|
| G31-3
|
8051539.67 |
-0.194846033 |
12 |
-2.338152396
|
| G32-1
|
17862328 |
0.7862328 |
12 |
9.4347936
|
| G32-2
|
16658101.7 |
0.66581017 |
12 |
7.98972204
|
| G32-3
|
16402476.3 |
0.64024763 |
12 |
7.68297156
|
PCR Results: Summary
- Our positive control PCR result was -8.127 μg/mL
- Our negative control PCR result was 2.802 μg/mL
Observed results
- Patient 69628: The photos for the drops for each patient were taken by a Samsung Galaxy S7 model camera app with the Selective Focus option in order to take the best resolution photos. The drops for G31-1 and G31-2 were clear and did not emit any fluorescence. The drop for G31-3 did have a subtle light-blue glow in the images from the LED. As for the qualitative aspect, two of the three calculations were more on the lower end of the spectrum which correlated with the negative control of 2.802 μg/mL. There was a slight fluctuation in the third calculation of about 12.35183484 μg/mL but could be due to experimental or human error from imaging. Since two out of the three results correlated most with the negative control it was concluded that the patient was negative for the disease SNP.
- Patient 57443: The drops were the same oblate spheroid shape as the drops from the first tested patient. The drops for G32-1, G32-2, and G32-3 also were clear and did not emit any fluorescence. There was still a subtle light-blue fluorescence due to the light from the LED. Comparatively, the qualitative portion of the experiment resulted in a range of results that are similar in range; respectively, 9.4347936 μg/mL, 7.98972204 μg/mL, and 7.68297156 μg/mL. Though the calculations are larger compared to the results of patient 69628, they also correlated most with the negative control that was calculated. Based on the observed results, it was concluded that the patient was negative for the disease SNP.
Conclusions
Positive control: -8.127 μg/mL
Negative control: 2.802 μg/mL
- Patient 69628: The results calculated from the derived images of 12.35183484 μg/mL, 0.629334 μg/mL, and -2.338152396 μg/mL were most closely related to the negative control of 2.802 μg/mL. Each of the calculations was most close to the negative control therefore based on the results of the replicates, the patient is most likely negative for disease SNP.
- Patient 57443: The results calculated from the derived images of 9.4347936 μg/mL, 7.98972204 μg/mL, and 7.68297156 μg/mL were also most closely related to the negative control of 2.802 μg/mL. Each of the calculations was most close to the negative control therefore based on the results of the replicates, the patient is most likely negative for disease SNP.
.
|
BME 100 Spring 2018
