The goal of this week is to accurately measure different concentrations of DNA using a fluorimeter and a smartphone.
PCR Reaction Report
Last week we set up our reaction tubes to run a polymerase chain reaction for two patient DNA samples. The pre-lab aided in showing us the proper micropipetting techniques, which resulted in more accurate micropippetting. We understood the differences between the first and second stops on the pipettor, with the first stop being used for drawing up the liquid and the second stop used to thoroughly dispel the liquid. The reactions did not have the same amount of liquid, as some of our sample material did not have 50 microliters in it or the sample was one of the first we set up (our skills were not perfect yet). There was liquid left in some of the tubes that had DNA samples and PCR reaction mix, as some of the tubes had more than 50 microliters or our skill were again not perfect. We did not have to change our labeling scheme.
Fluorimeter Procedure
Imaging set-up
We first used a box to set the fluorimeter up high enough to be seen by the smartphone. The fluorimeter was set on the box, and the smartphone stand set up 6 centimeter away. We then checked the camera of the smartphone, making sure that it could see the fluorimeter area that would contain the droplets. Once this was verified, a glass slide with spots to hold and stabilize the drops was secured onto the fluorimeter. A box with a swinging door was then placed over this setup to ensure that the image was light-free. We used the automatic settings on the phone's camera, with the flash turned off and a three-second timer turned on to make sure we could close the door of the box. ImageJ was then downloaded onto our laptop for use in diagnostics. The conditions of the fluorimeter image capture were maintained throughout this experiment.
Smartphone model: Google Pixel 2 XL
(ISO400, White balance auto, Exposure auto, Saturation auto, Contrast low)
Placing Samples onto the Fluorimeter
Set up fluorimeter stand described above, ensuring that the test area is dark, the glass slide is clean and appropriately placed, and that the camera takes clear images.
Obtain sample tray containing 8 red-dot tubes of 500 microliters buffer, 2 tubes of 1000 microliters SYBR Green 1 solution, 1 tube of 1000 microliters pH 8 water, and 5 concentration-labeled tubes of Calf Thymus DNA.
Pipette 160 microliters of water onto the glass slide (the drop should maintain its spherical shape) so that the fluorimeter light passes through it.
Turn on the fluorimeter, prepare camera, close door, and obtain image; make sure that this image is clear and focused, and that the fluorimeter light is focused through the drop. View video in lab folder for demonstration.
Repeat steps 3 and 4, using 80 microliters of SYBR Green I and 80 microliters of the different Calf Thymus DNA solutions (5, 2, 1, 0.5, 0.25, and 0). The total liquid of each drop (6 total) should equal 160 microliters.
Label each image with the appropriate concentration and send to laptop for analysis.
Repeat this process for DNA samples, making sure to keep consistent labels.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
0.5 μg/mL Calf Thymus DNA
0.5 μg/mL Calf Thymus DNA
H2O μg/mL Calf Thymus DNA
Note: these are full - color images, the images used for testing were isolated for green light and appeared in grey sccale. These images were used for qualitative comparison purposes.
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
5
2.5
C1
200418
2
1
C2
151413
1
0.5
C3
107331
0.5
0.25
C4
82909
0.25
0.125
C5
49334
0
0
C6
32151
Calibration curves
Images of Our PCR Negative and Positive Controls
Negative Sample
Positive Sample
PCR Results: PCR concentrations solved
PCR Product TUBE LABEL
RAWINTDEN Drop - background
PCR Product Concentration (µg /mL)
Total Dilution
Initial PCR Product Concentration (µg /mL)
G17 1-1
100410
0.673745113
12
8.084941355
G17 1-2
121350
1.003862404
12
12.04634885
G17 1-3
111350
0.846213268
12
10.15455921
G17 2-1
112160
0.858982848
12
10.30779417
G17 2-2
135220
1.222521756
12
14.67026107
G17 2-3
125920
1.075908059
12
12.91089671
G17 +
322100
4.168668811
12
50.02402573
G17 -
117100
0.936861521
12
11.24233825
PCR Results: Summary
Our positive control PCR result was 50.02 μg/mL
Our negative control PCR result was 11.24 μg/mL
Observed results
Patient 38060: These droplets all visually resembled those of the negative DNA control sample, with no green fluorescence seen. The average concentration was 10.10 μg/mL.
Patient 54209: These droplets all visually resembled those of the negative DNA control sample, with no green fluorescence seen. The average concentration was 12.63 μg/mL.
Conclusions
Patient 38060: The results were comparable to the negative control value (11.24 μg/mL), therefore drawing the conclusion that this patient does not have the SNP.
Patient 54209: The results were comparable to the negative control value (11.24 μg/mL), therefore drawing the conclusion that this patient does not have the SNP.
BME 100 Fall 2018
