The prelab reading was very helpful because it explained in depth how to pipette. We were confident when we began pipetting because of the simulations we did before class. We all understood the difference between the first and second stops. Before we began using the actual solutions, we practiced with the micropipettor using water. After we did our micropipetting, there was no noticeable difference in the amount of liquid in the tubes. There was no liquid left in the tubes that contained the DNA samples and the PCR reaction mix.
We did change our labeling scheme a little. We kept the G12 the same on all the tubes but then we labeled the tubes with patient ID 37106 with the labels 3-1, 3-2, and 3-3. We labeled the tubes with patient ID 54597 with the labels 5-1, 5-2, and 5-3.
Fluorimeter Procedure
Imaging set-up
In the light box, the flourimeter was placed on top of two boxes to prop it up to the height of the camera. We put the smartphone in the smartphone cradle exactly 8 cm from the light in the flourimeter. The flash was disabled on the camera. The ISO was set to the maximum and the exposure was set to +2.0. The timer on the camera was set for 10 seconds so we would have enough time to close the light box. This ensured that the pictures were always taken in darkness. We also made sure the picture was in focus both before and after each picture was taken. We also took three pictures of each new situation to ensure that they were consistent. See picture below for complete set up.
Placing Samples onto the Fluorimeter
Place slide in flourimeter with rough side up.
Using a micropipettor place 80 mircoliter of SYBR GREEN 1 on the slide between the first two rows. The drop should be round, balled shaped.
Add 80 microliters of a calf thymus solution to the SYBR GREEN 1 drop.
Move the slide so the light is in the middle of the drop.
Take a picture using the timer on the smartphone camera while the light box is covering the complete set up so as much light it removed as possible.
Take three focused pictures of the drop.
Remove the box being careful to keep the set up in place.
Use the micropipettor to remove the drop and discard the waste.
Repeat these sets for each concentration of the calf thymus solution.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA 5 μg/mL DNA sample
0.5 μg/mL DNA sample
zero DNA sample
Calibrator Mean Values
Initial Conc 2X Calf Thymus DNA (µg/mL)
Final DNA Conc SYBR Green I (µg/mL)
Sample Number
RAWINTDEN DROP - BACKGROUND (Three Images)
Mean
Standard Deviation
5
2.5
C-1
4142534 | 4140015 | 4114948
4123499
15251
2
1
C-2
5160256 | 5198834 | 5149773
5169621
25836.46355
1
0.5
C-3
5385062 | 5390132 | 5432693
5402629
26159.30613
0.5
0.25
C-4
3515080 | 3543942 | 3550231
3536417.667
18744.5964
0.25
0.125
C-5
4065530 | 4090712 | 4068633
4074958.333
13731.01097
0
0
C-6
1304992 | 1306585 | 1269108
1293561.667
21192.46971
Calibration curves
A third graph is included without the two highest concentrations in order to create a better calibration curve. The standard deviations are included on all three graphs, but they are very small and hardly visible because of the dots.
Images of Our PCR Negative and Positive Controls Positive control PCR sample
Negative control PCR sample
PCR Results: PCR concentrations solved
PCR Product TUBE LABEL
MEAN (of RAWINTDEN DROP - BACKGROUND)
PCR Product Concentration (µg /mL)
Total Dilution (µL)
Initial PCR Product Concentration
Positive
4619611.67
0.654902918
0.0833
0.054553413
Negative
1548873.67
-0.112781583
0.0833
-0.009394706
Patient 3-1
4840513
0.710128250
0.0833
0.059153683
Patient 3-2
1446287.67
-0.138428083
0.0833
-0.011531059
Patient 3-3
5441817
0.860454250
0.0833
0.071675839
Patient 5-1
5889107.67
0.972276918
0.0833
0.080990667
Patient 5-2
5234250.67
0.808562668
0.0833
0.06735327
Patient 5-3
5375391
0.843847750
0.0833
0.070292518
PCR Results: Summary
Our positive control PCR result was 0.055 μg/mL
Our negative control PCR result was -0.009 μg/mL
Observed results
Patient 37106: Two of the three samples were very bright and looked similar to the positive PCR result. The third image was darker and looked more like the negative PCR result. The initial PCR product concentrations were calculated to be 0.059 μg/mL, 0.072 μg/mL, and -0.011 μg/mL.
Patient 54597: All three samples were very bright and looked similar to the positive PCR result. The initial PCR product concentrations were calculated to be 0.081 μg/mL, 0.067 μg/mL, and 0.070 μg/mL.
Conclusions
Patient 37106: Two of this patient's values were close to the positive PCR result so we conclude that this patient is positive. The third value that was similar to the negative PCR result could have been an experimental error which gave incorrect results.
Patient 54597: All three trials of this patient were close to the positive PCR result so we conclude that this patient is positive.
BME 100 Fall 2016
