After obtaining the required materials to start the PCR reaction, the tubes were labeled with the positive and negative control and the patient trial number. We began by adding 50 microliters of the appropriate PCR reaction mix to each of the labeled tubes. Since the mixture already contained some of the chemicals required to run the reaction, all that we needed to add was the sample that was to be examined along with the primers (these were already mixed.) Between each sample that was added to the tubes, the pipette tip was thrown in the proper waste container and a new one was used for the next tube in order to avoid contamination. After adding the PCR mix to the tubes, the appropriate DNA sample was added into these same test tubes (the positive and negative control, the three replicates for patient 1, and the three replicates for patient 2.) A total of 50 microliters of the DNA/primer mix were added to the test tubes, making the total volume of the tubes being 100 microliters each. The tubes were then closed and placed in the thermocycler where the polymerase chain reaction would take place.
Overall, the team found the pipetting to be, although tedious, enjoyable and fairly easy. At first, the team struggled with making sure that all of the substance was picked up by the pipette, and by getting rid of all of it when dispensing it. After some practice, however, this got considerably easier. We realized that some of the substance was still inside the tip because we were not pushing the tip to the second stop. The pre-lab was considerably helpful for the team members that had never used a micropipette before and served as a useful refresher for the others with previous experience. For the second part of the lab, it was very difficult to perfectly line up the drop in between the two circles of the slide so that the light would shine through properly. It was also difficult to ensure that, once the drops were successfully placed on the tray, nothing was moved or bumped so the drop remained intact. The team did not have to change the labeling scheme, as none of our labels were confusing or nontransferable. The team found that there were samples leftover in both the DNA and PCR reaction mix, as previously mentioned because we were not pressing the second stop all the way. We did ensure, however, that the same amount of liquid was pipetted for each trial.
Fluorimeter Procedure
Imaging set-up
While setting up the fluorimeter for the pictures in the lightbox, the following procedures were carried out. First, a fluorimeter kit was collected. The kit included the fluorimeter, a black box, the glass slides and a phone stand. One glass slide was placed into the back of the fluorimeter, with the smooth side facing down. Two lids and a container were then used to elevate the fluorimeter so that it would line up with the camera view. This was to ensure that the camera would capture the drop from the side and to make sure that the drop was focused. The phone used for the experiment was an iPhone 7 Plus, with its flash settings turned off. The black box from the fluorimeter kit was used to create a darker environment for the experiment, and allow us to see if any of the samples that were being tested would glow green.
Placing Samples onto the Fluorimeter
1) Set the lightbox up and turn on the fluorimeter.
2) Make sure the slide is placed in the fluorimeter with the smooth side down.
3) Set the micropipette to 80 microliters.
4) Pipette 80 microliters of the SYBR Green 1 solution and drop it onto the middle of the 2 circles of the glass slide.
5) Remove the micropipette tip and replace it with a new tip.
6) Pipette 80 microliters of the calibration or DNA sample and drop it into the middle of the glass slide, were the existing drop is.
7) Set the micropipette aside.
8) Make sure the light illuminates the center of the drop and adjust the slide, if necessary.
9) Place the black box that came with the fluorimeter kit around the fluorimeter in order to create a dark environment.
10) Place the iPhone 7 Plus at about 4-5 centimeters away from the slide and use it to take three images of the drop, making sure the drop is in focus.
11) Once the pictures have been taken, pipette the mixture off of the glass slide and dispose of it in a waste beaker.
12) Repeat this process for several DNA samples, making sure to move back the glass slide as the experiment proceeds.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
PCR 0 calibration
PCR 1 calibration
PCR 5 calibration
Calibrator Mean Values
Table 1: Calibrator Raw Data
Sample Number
Image number
Final DNA concentration in SYBR Green I solution (µg/mL)
AREA
Mean Pixel Value
RAWINTDEN OF THE DROP
RAW INTDEN OF THE BACKGROUND
C-1
1
0.25
37136
101.642
3774573
233699
C-1
2
0.25
28648
154.399
4423214
163892
C-1
3
0.25
12456
189.242
2357196
74206
C-2
1
0.5
16724
150.162
2511310
97498
C-2
2
0.5
20008
134.508
2691233
165057
C-2
3
0.5
20008
8.25
165057
69683
C-3
1
1
13272
183.905
2440791
72639
C-3
2
1
10788
213.754
2305976
70972
C-3
3
1
9432
203.973
1923869
62233
C-4
1
2
15388
191.203
2942236
73944
C-4
2
2
18168
218.469
3969147
99600
C-4
3
2
17096
182.133
3113741
80449
C-5
1
5
10560
240.453
2539186
73422
C-5
2
5
21108
80.95
1708701
133028
C-5
3
5
21024
79.613
1673785
128724
C-6
1
0
0.035
193.433
141206
1884
C-6
2
0
36904
250.42
9241497
361111
C-6
3
0
36494
200.598
7320636
223640
Table 2: Mean Values and Standard Deviation
Initial Concentration of 2X Calf Thymus DNA solution (µg/mL)
Final DNA concentration in SYBR Green I solution (µg/mL)
Our positive control PCR result was 999.5593283 μg/mL.
Our negative control PCR result was 1785.13088 μg/mL.
Observed results
Patient 52396: In the mixture of the DNA sample of patient 52396 and the SYBR Green 1, no green pigment appeared. Quantitatively, this conclusion seems contradictory since the average value of Patient 52396's DNA concentration seems to be closer to those of the positive control.
Patient 37028: In the mixture of the DNA sample of patient 37028 and the SYBR Green 1, no green pigment appeared. Similarly to the other patient, the initial DNA concentration seems to be closer to the positive control although it does not show the expected physical properties.
Conclusions
Patient 52396: Positive; the average of the initial concentration for this patient is 1322.809524 micrograms per milliliter, which is closer to the positive control than the negative control. Although the patient may not show the "symptoms," he/she still seems to be carrying the disease.
Patient 37028: Inconclusive; two of the trials performed on the patient yielded results that were closer to the positive control. However, if we were to average the three initial concentrations taken from the three trials, this average (1438.190065 micrograms per milliliter), is closer to the negative control. This is due to the high concentration that was obtained from the third trial. In order to obtain more accurate results and not misdiagnose the patient, the best option would be to test the patient once more.
BME 100 Fall 2017
