OUR TEAM
LAB 5 WRITE-UP
PCR Reaction Report
Before attending lab, we read the pre-lab reading and completed the quiz. This prepareed us for pipetting the DNA samples and the PCR reaction mix. Each DNA sample, as well as the positive and negative control were added to each labelled PCR reaction tube. We labelled the tubes on the front side using a sharpie: P (positive), N (negative), 1 (patient 1), 2 (patient 2). We pipetted, drawing the liquid up using the first stop, and pushed the liquid out using the second stop. A new pipette tip was used for each sample. After they were all filled, we placed them in the OpenPCR.
Fluorimeter Procedure
Smart Phone Camera Settings
- Type of Smartphone: iPhone 6
- Flash: none
- ISO setting:2000
- White Balance: N/A
- Exposure:f/2.2
- Saturation:
- Contrast:
Camera set-up
Place the smartphone on a cradle. Then move it to a position so that its camera can see the slide from the edge on. Adjust height of fluorimeter if needed and make sure the camera's timer is set to 3 seconds. Once ready to take a picture of the drop on the slide, adjust the distance between the smartphone and the fluorimeter so that the camera is focused on the drop.
- Distance between the smart phone cradle and drop = 6 cm
Placing Samples onto the Fluorimeter
- Make sure the fluorimeter is on, the camera is set, and the smooth side of the slide is facing down. Place an 80 microliter drop of the SYBR Green I solution in the middle of the slide on the first two clear circles.
- Take an 80 microliter drop of the sample and place it on top of the SYBR Green I Solution. Make sure the light illuminates the drop's center.
- Cover the fluorimeter with the lightbox while leaving a flap up, make sure the camera is focused, press the timer button on the camera, and lower the flap.
- Take the drop from the slide and place it in the liquid waste container. Move the slide so that the light is in the center of the next two circles.
- Repeat these steps with all five positions on the slide and place slide in SHARPS container once done.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
5 μg/mL Sample
Error creating thumbnail: File missing
0.5 μg/mL Sample
Error creating thumbnail: File missing
0 μg/mL Sample
Error creating thumbnail: File missing
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 |
10937732 |
10937732 |
10937732 |
10937732 |
0
|
| 2 |
1 |
C-2 |
10479042 |
10479042 |
10479042 |
10479042 |
0
|
| 1 |
0.5 |
C-3 |
7564693 |
7564693 |
7564693 |
7564693 |
0
|
| 0.5 |
0.25 |
C-4 |
6349244 |
6349244 |
6349244 |
6349244 |
0
|
| 0.25 |
0.125 |
C-5 |
4797928 |
4797928 |
4797928 |
4797928 |
0
|
| 0 |
0 |
C-6 |
4101748 |
4101748 |
4101748 |
4101748 |
0
|
|
|
Calibration curves
Images of Our PCR Negative and Positive Controls
Positive Control
Error creating thumbnail: File missing
Negative Control
Error creating thumbnail: File missing
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)
|
|
| G11 + |
14058421.3 |
3.3528071 |
12 |
40.2336852
|
| G11- |
2672211.67 |
-0.44259611 |
12 |
-5.31115332
|
| G11 1-1 |
11292665 |
2.430888333 |
12 |
29.17066
|
| G11 1-2 |
11678217 |
2.559405667 |
12 |
30.712868
|
| G11 1-3 |
13581887 |
3.193962333 |
12 |
38.327548
|
| G11 2-1 |
2827443 |
-0.390852333 |
12 |
-4.690227996
|
| G11 2-2 |
3239641 |
-0.253453 |
12 |
-3.041436
|
| G11 2-3 |
2486281 |
-0.504573 |
12 |
-6.054876
|
|
|
PCR Results: Summary
- Our positive control PCR result was 40.2336852 μg/mL
- Our negative control PCR result was -5.31115332 μg/mL
Observed results
- Patient 24467 : The images that were taken of this patients DNA were fluorescent green. It was fairly easy to observe the green color within the drop. The quantitative results are 29.17066, 30.712868, and 38.327548 μg/mL for product tubes 1-1, 1-2, and 1-3 respectively.
- Patient 87309 : There was no visible color on the images taken of this patients DNA. The quantitative results are -4.690227996, -3.041436, and -6.054876 μg/mL for the product tubes 2-1, 2-2, and 2-3 respectively.
Conclusions
- Patient 24467 : In conclusion, patient 1 is positive due to the observed color and the overall product concentration values being very close to that of the positive thresh hold and very far away from the negative thresh hold.
- Patient 87309 :In conclusion, patient 2 is negative due to the lack of observable color and the overall product concentration levels being very close to that of the negative thresh hold and very far away from the positive thresh hold.
|
BME 100 Fall 2015
