OUR TEAM
LAB 5 WRITE-UP
PCR Reaction Report
The micropipetting lab went relatively smoothly for our group and we finished fairly early due to our prior understanding of the concepts we used in class. The pre-lab videos and interactive games helped illustrate how to use the micropipettor. It showed us step by step how to properly use it and how to make sure our measurements were as accurate as possible. The first stop on the micropipettor is the way to get the proper amount of liquid into the device. The second stop is used when ejecting the liquid into the desired location because sometimes excess liquid is left behind after the first stop and the second stop takes care of this problem. The final reactions did have exactly the same amount of liquid because we accurately set the micropipettor. There was extra liquid in the tubes that the DNA samples and PCR reaction mix because we were given excess amounts to properly measure out the correct amount of each liquid. We did not have to change our labeling scheme. We kept it the same. Overall, our group's experience with the micropipetting lab went well.
Fluorimeter Procedure
Smart Phone Camera Settings
- Type of Smartphone: Iphone 6
- Flash: Off
- ISO setting: N/A
- White Balance: N/A
- Exposure: N/A
- Saturation: N/A
- Contrast: N/A
Camera set-up
We set up the camera such that the lens was at the same height as the drop we then braced the phone so we would not have any tilt. We made sure that the camera will not budge. We made sure we had the most stability we could because focusing was a major issue.
We set the distance between the smart phone cradle and drop to be 5.25 cm because 4 cm was just too close to focus and get clear pictures.
- Distance between the smart phone cradle and drop = 5.25 cm
Placing Samples onto the Fluorimeter
- Make sure the black box is set up so that outside light can be adequately blocked and that the fluorimeter is steady.
- Place a slide in the fluorimeter, making sure that the rough side of the slide is face up.
- Set the micropipettor to 80 microliters.
- Use the micropipettor to add 80 microliters of the SYBR GREEN I to the rough side of the slide in the middle of the dots.
- Add 80 microliters of the sample onto the slide on top of the drop of SYBR GREEN I.
- Adjust the slide so that the center of the drop is lit up by the light in the fluorimeter.
- Place the camera phone in the cradle and make sure that it is steady and focused on the drop.
- Take three pictures of the drop using a timer and make sure that as little light as possible enters the box.
- Remove and dispose of the drop and shift the slide to its next position.
- Repeat steps 4 through 9 with each different sample (do not move the camera phone from its position).
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
0 DNA
Error creating thumbnail: File missing
0.5 μg/mL sample
Error creating thumbnail: File missing
5 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
|
|
|
|
Image 1 |
Image 2 |
Image 3 |
|
|
| 5 |
2.5 |
C-1 |
23240116 |
22421317 |
24789068 |
23483500.33 |
1202492.474
|
| 2 |
1 |
C-2 |
11883245 |
12100913 |
11923181 |
11969113 |
115875.5842
|
| 1 |
0.5 |
C-3 |
5344939 |
5794711 |
2990034 |
4709894.667 |
1506324.748
|
| 0.5 |
0.25 |
C-4 |
2130311 |
9313859 |
1255836 |
4233335.333 |
4421534.482
|
| 0.25 |
0.125 |
C-5 |
16459474 |
10749653 |
2597881 |
9935669.333 |
6966553.465
|
| 0 |
0 |
C-6 |
8791812 |
8872852 |
8460202 |
8708288.667 |
218636.9503
|
Calibration curves
Error creating thumbnail: File missing
Error creating thumbnail: File missing
Images of Our PCR Negative and Positive Controls
Negative control: Error creating thumbnail: File missing Positive 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)
|
Total Dilution
|
Initial PCR Product Concentration (µg /mL)
|
"
|
| G13 + |
2729508 |
-4.270492 |
12 |
-51.245904
|
| G13 - |
1401920.333 |
-5.59807 |
12 |
-67.17684
|
| G13 1-1 |
2958099.667 |
-4.0419 |
12 |
-48.5028
|
| G13 1-2 |
3262745.667 |
-3.7372543 |
12 |
-44.8470516
|
| G13 1-3 |
3490186 |
-3.509814 |
12 |
-42.117768
|
| G13 2-1 |
1219826.667 |
-5.780173333 |
12 |
-69.36208
|
| G13 2-2 |
1425015 |
-5.574985 |
12 |
-66.89982
|
| G13 2-3 |
1451893.333 |
-5.548106667 |
12 |
-66.57728
|
|
|
PCR Results: Summary
- Our positive control PCR result was -4.270492 μg/mL
- Our negative control PCR result was -5.59807 μg/mL
Observed results
- Patient 21939 : These images showed the droplet was very white looking, similar to the positive control.
- Patient 73606 : These images showed the droplet was clear and revealed the dark background behind it, similar to the negative control.
Conclusions
- Patient 21939 : This patient turned out to be positive. We arrived at this decision because the droplet looked more similar to that of the positive control.
- Patient 73606 : Patient 73606 was negative and this was due to the extreme similarities between the negative control droplet's appearance and the patient's droplet.
| 
BME 100 Fall 2015
