Our group's pipetting experience was fairly easy and successful. By understanding the readings given to us prior to the lab detailing the procedure to most efficiently pipette the samples, even the members of the group who had never pipetted before were able to do so easily. We understood that the first stop was used so that we could suck the liquid into the pipettor and the second stop was used to eject the liquid out once again. Despite our pipetting being successful, all of the final reactions did not have exactly the same amount of liquid. Even though we tried to be precise in our measurements, we could not be exact sometimes because the liquid would sometimes get stuck in the tips of the micropipettors despite our best attempts to eject it completely. Also, some liquid would remain in the test tubes. However, despite these inexact amounts of liquids, the process was successful as a whole because these things were accounted for in the procedure. We did not have to change our labeling scheme because it was detailed enough as is for us to understand what we were doing and what was in each tube. Overall, the experience was enlightening and the process ran very smoothly.
Fluorimeter Procedure
Imaging set-up
We took our images with the equipment in that came in the black fluorimeter box: the reversible box, the fluorimeter, and the phone stand. We turned the box over and unbuttoned the side to create a natural cover for the fluorimeter (by resting the side against a lab chair). We then placed an iPhone 6 inside the phone stand and used various miscellaneous items to get the fluorimeter to the level of the iPhone camera. To get the best results, we only pressed the capture button after we could confirm the camera focused in the dark and took 3 pictures.
Placing Samples onto the Fluorimeter
1. Make sure the fluorimeter is turned on. The flourimeter should have an LED lighted up and the metallic switch on the side should be flipped if the LED isn't lighted up to turn it on.
2. Find the smooth side of a slide by rubbing it with your gloves and slide it into the fluorimeter smooth side down.
3. Micropipette 80 uL of SYBR Green I solution on the two clear circles in the middle of the first row.
4. Micropipette 80 uL of the tested solution on the two clear circles in the middle of the second row. You should be using a fresh tip and the two drops should merge.
5. Place the fluorimeter atop whatever items needed to have it level with the camera being used.
6. Make sure the camera is focused and take as many pictures as needed.
7. Repeat steps 1-6 for all necessary samples.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
Figure 1: An ImageJ photo for a 5 μg/mL (High) calibrated sample.
Figure 2: An ImageJ photo for a 0.5 μg/mL (Low) calibrated sample.
Figure 3: An ImageJ photo for a 0 μg/mL calibrated sample.
Calibrator Mean Values
Initial Concentration of 2X Calf Thymus DNA solution (µg/mL)
Final Concentration DNA concentration in SYBR Green I solution (µg/mL)
Sample #
RAWINTDEN DROP-BACK: Image 1
RAWINTDEN DROP-BACK: Image 2
RAWINTDEN DROP-BACK: Image 3
MEAN
Standard Deviation
5
2.5
C-1
15042226
13415220
14660133
14372526.33
850779.247
2
1
C-2
9861671
9749216
9435037
9681974.667
221122.5682
1
0.5
C-3
11511409
10179808
11178423
10956546.67
692973.4023
0.5
0.25
C-4
5949334
6037283
5798961
5928526
120515.8644
0.25
0.125
C-5
6033893
6688534
6412014
6378147
328631.9217
0
0
C-6
4621433
4931946
5627734
5060371
515296.1878
Calibration curves
Images of Our PCR Negative and Positive Controls
Figure 4: An ImageJ photo for our negative control of Calf Thymus DNA (μg/mL)
Figure 5: An ImageJ photo for our positive control of Calf Thymus DNA (μg/mL)
PCR Results: PCR concentrations solved
PCR Product TUBE LABEL
MEAN (of RAWINTEN DROP-BACKGROUND)
PCR Product Concentration (µg/mL)
Total Dilution
Initial PCR Product Concentration (µg/mL)
G12+
8460807
0.820269
12
9.843228
G12-
3028743
-0.990419
12
-11.885028
G12 1-1
3885115
-0.704961667
12
-8.45954
G12 1-2
3158441
-0.947186333
12
-11.366236
G12 1-3
4851679
-0.382773667
12
-4.593284
G12 2-1
4121757
-0.626081
12
-7.512972
G12 2-2
5351361
-0.216213
12
-2.594556
G12 2-3
3628793
-0.790402333
12
-9.484828
PCR Results: Summary
Our positive control PCR result was 0.820269 μg/mL
Our negative control PCR result was -0.990419 μg/mL
Observed results
Patient 76650: The three samples appeared to vary in florescence; one sample displayed a relatively weak glow that looked similar to the appearance of our 0.25 μg/mL calibrated sample. The second sample displayed a very clear green florescence, and the third sample displayed an extremely faint green florescence in the middle of the droplet. Surprisingly, the sample with the relatively weak glow was measured to have a concentration of -0.704961667 μg/mL. The sample with the strongest display of florescence had a measured concentration of -0.947186333 μg/mL, and the sample with the faintest florescence had a concentration of -0.382773667μg/mL.
Patient 80605: All three samples displayed a weak misty green florescence towards the middle of the droplet. The calculated concentrations of the three samples for Patient 80605 are -0.626081μg/mL, -0.216213μg/mL, and -0.790402333μg/mL.
Conclusions
Patient 76650: As calculated from this lab's results, Patient 76650's average PCR product concentration was -0.6783072223 μg/mL. Compared to the positive and negative control values, Patient 76650's value was closest to our negative PCR result (which was -0.990419 μg/mL). However, 2 out of the 3 samples taken from Patient 76650 displayed detectable florescence under the fluorimeter, which is a physical indication of a positive sample. Thus, Patient 76650's results were inconclusive. Error in our set up or our calculations in ImageJ may have attributed to an inaccurate value for our positive control.
Patient 80605: As calculated from this lab's results, Patient 80605's average PCR product concentration was -0.544232111 μg/mL. Compared to the positive and negative control values, Patient 80605's value was closest to our negative PCR result (which was -0.990419 μg/mL). While there were faint traces of green fluorescence in Patient 80605's samples, the degree of the fluorescence's brightness was relatively consistent across the three samples and so this faint fluorescence may have been a result of slight contamination during our set up. Thus, Patient 80605's samples are negative.
BME 100 Fall 2017
