Preparation
While preparing the solutions for the PCR reaction, our group did a good job of staying focused and sharing the responsibilities. The pre-lab readings were helpful, but in the lab, not too practical. The use of the pipettor was very difficult for all members, and we had to try multiple times in some cases to get the liquid into the sample vials. The difference between and the use of the multiple stops was not clear to some members and were a hurdle that was very large. In one case, a member could not get much of the solution out of its original vial and the final reaction did not have the same amount of liquid as all the rest. In some cases, there was residual liquid left in the original vials and may cause some error in the PCR and gel electrophoresis in the next section of the lab. The labeling scheme was consistent with that set up in Lab 4 and was not changed. Overall, practice trials or going over the pre-lab video about how to use the micropipette in class with instructions and details from the professor would have been much more helpful in the long run.
Fluorimeter Procedure
Imaging set-up
Set your camera's timer to three seconds.
Adjust the height of the fluorimeter, so that the drop is the same level as your camera.
Adjust the distance between the slide and your smartphone camera, so that the camera focuses more on the drop. The distance should be >4cm and record the distance.
Put the black box around the fluorimeter and have one flap open. Lower the flap before you take the picture.
Placing Samples onto the Fluorimeter
Place the slide, rough slide down, into the fluorimeter.
Turn on the fluorimeter.
Place the 80 uL SYBR Green 1 solution on the first two dots in the middle of the slide, using the micropipette.
Then place the 80 uL of the PCR or calibration solution onto the drop previously placed.
Make sure that the light is through the drop of solution and adjust the slide as needed for this part.
After imaging, use the micropipette to suck up the solution and adjust the slide to the next set of dots.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
Zero Calf Thymus DNA (H2O Calibration)
Low Calf Thymus DNA (0.5 Calibration)
High Calf Thymus DNA (5 Calibration)
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
13177890
14435223
15559490
14390867.67
1191419.4
2
1
C-2
12300321
12632424
13015413
12649386
357847.6271
1
0.5
C-3
9046300
11059099
12621105
10908834.67
1792133.428
0.5
0.25
C-4
4672426
4753979
5059973
4828792.667
204318.337
0.25
0.125
C-5
7824846
4854867
5218871
5966194.667
1619896.081
0
0
C-6
4387479
4556983
3112914
4019125.333
789365.0214
Calibration curves
Images of Our PCR Negative and Positive Controls
PCR Results: PCR concentrations solved
PCR Product TUBE LABEL
MEAN RAWINTDEN
PCR Product Concentration (µg /mL)
Total Dilution
Initial PCR Product Concentration (µg /mL)
+
12411750
2.1029375
12
25.23525
-
4596242.333
0.149060583
12
1.788727
g1-1
12318643
2.07966075
12
24.955929
g1-2
12725884.33
2.181471083
12
26.177653
g1-3
13998751
2.49968775
12
29.996253
g2-1
3823428
-0.044143
12
-0.529716
g2-2
2455710.667
-0.386072333
12
-4.632868
g2-3
2814304.667
-0.296423833
12
-3.557086
PCR Results: Summary
Control results
Our positive control PCR result was 25.23525 μg/mL
Our negative control PCR result was 1.788727 μg/mL
Observed results
Patient 38066 : Most droplets for patient 1 were showing green glows even before they were put in full darkness, and the images came back with bright green fluorescent colors showing up. The final results were 24.96, 26.18, and 30.00 μg/mL.
Patient 66318 : Patient 2's photos were not green and did not show barely any green in the droplet. Any green in the photos could easily be traced back to errors with contamination on the slide itself. Its values were -0.53, -4.63, and -3.56 μg/mL.
Conclusions
Patient 38066 : Positive. The values of this patient were consistently close to that of the positive control leading to this conclusion.
Patient 66318 : Negative. The patient's values were nowhere near the positive control values, and also were very similar to the negative control.
BME 100 Spring 2018
