To prepare for this lab, the pre-labs were completed. They were helpful and allowed for each team member to learn how to properly use a pipettor and understand the difference between the first and second stop on the device.
The following steps were taken to set up the reaction:
Empty PCR tubes were cut in half (two strips of four linked tubes).
The sides of the empty tubes were labeled with the individual tube labels that our team created.
The tubes were then placed on a rack.
50 microliters were pipetted into the empty tube labeled positive control.
Using a different pipette tip, positive control DNA/primer mix were transferred into the same tube. After, the total volume in this tube was 100 microliters.
Steps 4 and 5 were repeated for the negative control, patient 1 replicates 1, 2, and 3, and patient 2 replicates 1, 2, and 3. The appropriate DNA/primer mixes were used for each corresponding tube.
The tubes were taken over to our group's assigned PCR machine and placed into the slots in the heating block.
The final reactions did not have the exact same amount of liquid; there were slight differences. There was still liquid left in the tubes that had the DNA samples and PCR reaction mixes. Our group did not have to change our labeling scheme.
Fluorimeter Procedure
Imaging set-up
A portable light box, which enhanced the detection of fluorescence, was used to take pictures in the dark. A stand was used to prop up the smartphone that was used to take the pictures. On the phone's camera, the timer was set for three seconds and flash was turned off. The height of the fluorimeter was adjusted as needed to get a camera view of the slide nearly edge-on. The distance between the phone and the fluorimeter was also adjusted to where the drop could be viewed closely but still be in focus. Before taking the picture, the lower flap of the light box was lowered.
Placing Samples onto the Fluorimeter
After putting on gloves, the "smooth" side of a slide was found.
The fluorimeter was then turned on and placed on a table.
The slide was then placed in the fluorimeter with the smooth side down.
A 80 microliter drop of SYBR Green I solution was placed on the first two clear circles in the middle of the slide.
Next, a 80 microliter drop of SAMPLE/CALIBRATION solution was placed on top of the SYBR Green I drop.
The slide was adjusted in such a way that the light would illuminate the center of the drop and the drop would focus the light on the other side.
The fluorimeter was covered with the lightbox and prepared for pictures.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
Calibrator Mean Values
Initial Concentration (micrograms/mL)
Final DNA Concentration (micrograms/mL)
Sample Number
Image 1 Drop-Background
Image 2 Drop-Background
Image 3 Drop-Background
Mean
Std Dev
5
2.5
C-1
389463
430724
373986
398057.6667
29329.18994
2
1
C-2
309203
392743
361095
354347
42176.82454
1
0.5
C-3
389800
369876
376190
378622
10182.21057
0.5
0.25
C-4
333932
307776
347263
329657
20087.62084
0.25
0.125
C-5
284095
279540
294893
286176
7885.212299
0
0
C-6
72831
86503
80357
79897
6847.597827
Calibration curves
Images of Our PCR Negative and Positive Controls
PCR Results: PCR concentrations solved
PCR Product Tube Label
Mean
PCR Product Concentration (micrograms/mL)
Total Dilution
Inital PCR Product Concentration (micrograms/mL)
P
310827.333
-129.667
12
-1556.004
1-1
87918.66667
-223038.333
12
-2676460
1-2
142840
0.19979
12
2.39748
1-3
167730.6667
0.318265
12
3.819183
N
143833.6667
0.204519
12
2.45423
2-1
249175.6667
0.70593
12
8.47117
2-2
273149.3333
0.82004
12
9.8405
2-3
150892
0.238116
12
2.85739
PCR Results: Summary
Our positive control PCR result was -1556.004 μg/mL
Our negative control PCR result was 2.45423 μg/mL
Observed results
Patient 19974 : Negative. There was not any light reflected in any of the pictures of the data. The sample was remained dark like the negative control
Patient 17269 : Positive. The image had a green light reflected in two of the samples. The samples were bright and similar to the positive control.
Conclusions
Patient 19974 :Inconclusive. The value for this patient was a large negative number for the first sample , just like the positive control. However, the number was a larger negative number than the positive control. The other two samples gave results similar to negative control.There does not seem to be a definitive trend in the sample's results, making the results inconclusive.
Patient 17269 :Negative. The value for this patient was a small positive number for all of the different samples, like the negative control.
Extra Credit Gel Electrophoresis Experiment
Image I: Results of Group eight's electrophoresis experiment:
Conclusion/Results:
Gel Electrophoresis is used in labs to separate charged molecules, like D.N.A, R.N.A and Proteins, according to their size and charge. An electric field is generated by a power supply that moves the negatively charged D.N.A from the positive side of the gel to the negative side of the gel until the Electrophoresis Retardation force and friction force of the D.N.A molecules are great enough to stop the force on the D.N.A molecules by the electric field. The less massive and less charged the D.N.A, the farther the D.N.A goes. The Latter, on the far left column of image I, was used as a base to compare the samples and to confirm that the Gel Electrophoresis process is working correctly. The positive control was put into the second column from the left and the negative control was put in the sixth column from the left. Notice that the positive control goes as far down as the lowest point in the ladder, while the negative control doesn't show any reaction. In this Electrophoresis experiment, Patient one tested negative in all three samples while Patient two tested positive in all three samples. These results suggest that Patient one is negative for the D.N.A in question and that Patient two is positive for the D.N.A in question.
BME 100 Spring 2017
