Name: Sebastian Tellez Role: Innovative Researcher
LAB 5 WRITE-UP
PCR Reaction Report
Since we were rather new at micropipetting, all team members contributed to micropipetting the samples so we could all gain that experience. We were all able to pipette without too much issue. The pre-lab reading and instructional video proved instrumental for us to complete this part of the lab. With this prior knowledge, we were able to pipette correctly and verify that all group members were pipetting correctly as well. Before we used the pipette, we each tested the micropipettor to identify the first and second stop. This way we would be confident when actually pipetting the solution. The final reactions had a similar amount of liquid, but some tubes had slightly more liquid than other. We attributed this to minor error in pipette technique such as: releasing from the pipette stop too quickly and getting air bubbles. There was no liquid left in the tubes, but there is possibility that small amounts, not visible to the eye, remained. We did not have to change our initial labeling scheme.
The pipetting section went smoothly and the tubes were then ready to be placed in the OpenPCR machine. Once we had another group to share the machine, the settings were marked and the machine was closed and started. After the cycles were complete, the samples were preserved in the freezer until our next lab.
Fluorimeter Procedure
Imaging set-up
1. Identify a smartphone camera to take pictures with (we used a Samsung phone) and set the recommended camera settings:
Inactivate the flash
Set ISO to 800 or higher
Set white balance to auto
Set exposure to highest setting
Set saturation to highest setting
Set contrast to lowest setting
2. Place the phone in the cradle
3. Adjust height of fluorimeter so that the camera aligns with the slide.
We added three trays to the bottom of the fluorimeter and placed the smartphone 5.0 cm away from the fluorimeter so the images would be close enough to the sample and keep the image in focus.
4. Set the camera timer to three seconds to allow ample time to close the lightbox lid for the picture.
Placing Samples onto the Fluorimeter
1. Insert a slide in the fluorimeter with the smooth glass side down
2. Turn on the excitation/blue LED light.
3. Use a micropipette to drop 80 microliters of SYBR Green I in between the first two dots of the slide and is centered with the excitation light.
4. Micropipette an 80 microliter drop of the sample/calibration solution on top of the drop of SYBR Green I.
5. Start camera timer and lower lid of the lightbox in time for the picture.
6. Repeat step 6 for a total of three pictures.
7. Open lid and micropipette sample and dispose in the designated waste container
8. Move the slide to the next position for a new sample.
9. Repeat from step 3-8 for all other samples.
Next Step
Use ImageJ to analyze these images and draw a final conclusion for each patient.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
High: 5 μg/mL sample'
Low: 0.5 μg/mL sample
0 μg/mL sample
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
4542918
4558634
4520038
4540530
19408.49587
2
1
C-2
5384193
5438443
5424641
5415759
28194.55742
1
0.5
C-3
3646884
3564298
3614559
3608580.333
41616.34523
0.5
0.25
C-4
4824435
4838666
4814922
4826007.667
11949.86796
0.25
0.125
C-5
3887120
4014102
4164017
4021746.333
138606.6882
0
0
C-6
1300777
1293316
1305037
1299710
5932.902072
Calibration curves includes all data points
without highest data point value
Images of Our PCR Negative and Positive Controls
Negative Control
Positive Control
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)
G7 +
5262558.667
2.262558667
12
27.150704
G7 -
846462
-2.153538
12
-25.842456
G7 1-1
5478185.667
2.478185667
12
29.738228
G7 1-2
3847307.667
0.847307667
12
10.167692
G7 1-3
5144280.667
2.144280667
12
25.731368
G7 2-1
2077352.333
-0.922647667
12
-11.071772
G7 2-2
1422916.667
-1.577083333
12
-18.925
G7 2-3
1623531.333
-1.376468667
12
-16.517624
PCR Results: Summary
Our positive control PCR result was 27.15 μg/mL.
Our negative control PCR result was -25.84 μg/mL.
Observed results
Patient 28063 : The image has a greener tint, hinting that there is a statistically significant amount of DNA present in the solution. The three samples for patient 28063 reflects the qualitative conclusion and is most similar to the positive control. Each trial has the following quantities in μg/mL: (1) 29.74, (2) 10.16, and (3) 25.73.
Patient 28963 Sample 1
Patient 67655 : The image lacks much of a green tint, hinting that there is not a statistically significant amount of DNA present in the solution. The three samples for patient 67655 reflects the qualitative conclusion and is most similar to the negative control Each trial has the following quantities in μg/mL: (1) -11.07, (2) -18.93, and (3) -16.52.
Patient 67655 Sample 2
Conclusions
Patient 28063 : Each sample for patient 28063 is most similar to the positive control of 27.15 μg/mL. The average of the three samples is 21.88 μg/mL. Because this value is very similar to the positive control, one can conclude that patient 28063 is the positive control.
Patient 67655 : Each sample for patient two is most similar to the negative control of -25.84 μg/mL. The average of the three samples is -15.51 μg/mL. Because this value is most similar to the negative control, one can conclude that patient 67655 is the negative control.
BME 100 Fall 2018
