While none of our group members had used a micropipettor previously, the pre-lab reading was helpful and we encountered few issues. The first stop of the pipettor was used to draw the liquid into the instrument and the second stop was used to release all the liquid. The final reactions had approximately the same amount of liquid, but not exactly. Although each tube should have had exactly 100 microliters of liquid total, the tubes we were given to start with contained 51 microliters of PCR mix and 51 microliters of each DNA sample. For a few of the tubes, an error was made and not all 50 microliters were drawn into the pipette on the first try. In order to remedy the problem, the micropipette was used a second time to draw in the rest of the liquid since we knew that the tube contained only the amount we needed to use. Ultimately, the result was that some of the DNA sample and PCR reaction mix tubes were completely empty at the end, and some contained a minute amount of liquid. Consequently, the final reactions had approximately the same amount of liquid (within 2 microliters).
We were able to use the same labeling scheme as described in our previous write-up, and although we encountered a few bumps, overall the micropipetting process proceeded smoothly.
Fluorimeter Procedure
Imaging set-up
The camera of a Samsung 8 smartphone was set up with an inactive flash, iso set to 800 or higher, white balance set to auto, exposure and saturation set to the highest setting, and contrast set to the lowest setting. Once the camera was set up, a three second timer was turned on, and the phone was placed on the cradle at a right angle from the slide. The height of the fluorimeter and slide was then adjusted so that the phone camera could take a picture of the drop sideways edge-on. The distance between the phone and the fluorimeter was measured and recorded (it had to be at least four centimeters away).
Placing Samples onto the Fluorimeter
Place the fluorimeter on the table and turn it on.
With gloves on find the side of slide with the smooth glass back. Put the slide in the fluorimeter with the smooth slide facing down.
Place smartphone in the cradle with a camera timer set for three seconds. Then, if needed adjust the height of the fluorimeter. The camera view needs to be of the slide nearly edge-on.
In the middle of the glass slide on the first two clear circles, place an 80 microliter drop of SYBR green I solution.
Place an 80 microliter drop of the sample solution on to of the drop of SYBR green I.
Move the slide so the light goes through the drop and focuses on the opposite side.
Keeping the drop in focus, adjust the distance between the smartphone and the fluorimeter. Measure and record the distance between them. (Note: The distance needs to be greater than four centimeters.)
Keeping one flap up, move the lightbox over the fluorimeter. Make sure the drop is focuses on the camera one more time.
Start the timer on the camera, before the picture is taken lower the flap.
Open lightbox carefully and remove all 160 microliters of liquid on the slide and discard the liquid.
Move the slide to the center of the next two circles and repeat steps 3-11.
There are 5 measurement positions per slide. If there are more sample solutions than measurement positions continue on a new slide starting with step 1.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
5 μg/mL Sample of Calf Thymus
0.5 μg/mL Sample of Calf Thymus
0 μg/mL Sample of Calf Thymus
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
21602954
19645209
21357610
20868591
1066558.043
2
1
C-2
23727069
16024843
21153788
20301900
3921142.208
1
0.5
C-3
14394113
22936740
15966091
17765648
4546748.625
0.5
0.25
C-4
18194498
16723814
17515761
17478024.33
736067.863
0.25
0.125
C-5
8995531
9089814
8673046
8919463.667
218548.7837
0
0
C-6
128313
86145
-14798
66553.33333
73539.54468
Calibration curves
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)
G11 +
19862657
1.607832125
12
19.2939855
G11 -
3632246.7
-0.420969163
12
-5.05162995
G11 1-1
4561694
-0.30478825
12
-3.657459
G11 1-2
4738982.3
-0.282627213
12
-3.39152655
G11 1-3
3273955.7
-0.465755538
12
-5.58906645
G11 2-1
15308738
1.03859225
12
12.463107
G11 2-2
20157686
1.64471075
12
19.736529
G11 2-3
17206181
1.275772625
12
15.3092715
PCR Results: Summary
Our positive control PCR result was 19.29 μg/mL
Our negative control PCR result was -5.05 μg/mL
Observed results
Patient 69222: From the images taken of Patient 69222, there seemed to be minimal green coloring in the droplet. Most of the drop was clear with just a hint of a clouded green color near the very bottom of the droplet. With just a line of deep green, Patient 69222's droplet closely resembled the droplet of the negative control. The similarity in colors between the droplets of Patient 69222 and the negative control gave an early indication as to which sample contained the disease marker. To quantitavely back the findings observed, the concentrations of light in each droplet were found. The calculated concentrations from Patient 69222 for three tirals were -0.305 μg/mL, -0.283 μg/mL and -0.466 μg/mL. All of the concentrations were considerably close to -0.421 μg/mL which was the concentration of the negative control. From the lack of green fluorescene displayed in the images and from the calculations, it became clear that Patient 69222 closely resembled the characteristics of the negative control.
Patient 45886: From the images captured of Patient 45886, the green fluorescense of the droplets shone brightly. The droplets from the various trials had a unifrom green glow throughout, unmistakably resembling the green fluorescense of the positive control. The fluorescence of the droplets from Patient 45886 gave a clue as to which sample contained the disease marker. The measured concentrations of the value of the fluorescence in each droplet also supported the similarity between Patient 45886 and the positive control. The droplets from Patient 45886 had concentration values of 1.04 μg/mL, 1.64 μg/mL and 1.28 μg/mL. These measurements were very close to the concentration of the positive control which was determined to be 1.61 μg/mL. Due to the proximity in concentration values and the resemblance in color, Patient 45886 drew strong comparisons among it and the postive control.
Conclusions
Patient 69222 : All 3 replicates of Patient 69222's PCR test yielded values (i.e. -3.66, -3.39, -5.59) closer to the negative control threshold value of -5.05. Ultimately, it was concluded that the patient was negative because of these results. Patient 69222 does not have the SNP rs1044498.
Patient 45886 : All 3 replicates of Patient 45886's PCR test yielded values (i.e. 12.46, 19.74, 15.31) closer to the positive control threshold value of 19.29. Ultimately, it was concluded that the patient was positive because of these results. Patient 45886 does have the SNP rs1044498.
BME 100 Spring 2018
