Our team already had a solid understanding of proper pipetting technique, but we appreciated the pre-lab reading. We understood the difference between the first stop (used to pipette up) and the second stop (used to release the liquid). The final reactions had the exact same amount of liquid (100 uL). There was maybe 20 uL of liquid left in the PCR mix tubes after taking our sample. We did not have to change our labeling scheme, because we came prepared.
Fluorimeter Procedure
Imaging set-up
We placed our device in the provided “phone holder” that was found in the dark box. The bottom of the phone was placed into the receiving end of the phone holder. The phone was further supported by the pop socket that was previously adhered to the back of the device. We set a 3 second timer and quickly closed the end of the box.
Placing Samples onto the Fluorimeter
Step 1: Place a slide on the fluorimeter.
Step 2: Pipette 80 uL of DNA onto the center circle of the slide closest to our image capturing device.
Step 3: Pipette 80 uL of Sybr Green onto the center circle of the slide immediately behind our DNA drop.
Step 4: Close the black box cover and turn on the fluorimeter.
Step 5: Take a picture with our phone using the timer and upload the photo to the computer to analyze with ImageJ.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
Water:
.5 μg/mL:
5 μg/mL:
Calibrator Mean Values
Table 2: Calibrator means
RAWINTDEN DROP-BACKGROUND
Initial Concentration of 2X Cell Thymus DNA solution (microgram/mL)
Final DNA concentration in SYBR green 1 solution(ug/mL)
Sample Number
Image 1
Image 2
Image 3
MEAN
STANDARD DEVIATION
5
2.5
c-5
16893535
18925140
19464765
18427813.33
1355841.542
2
1
c-2
20813999
19425623
18876021
19705214.33
998783.3436
1
0.5
c-1
21312146
19883957
19310769
20168957.33
1030677.625
0.5
0.25
c-0.5
18508803
18894005
18200885
18534564.33
347277.3651
0.25
0.125
c-0.25
20221523
21565488
20831981
20872997.33
672920.6751
0
0
H2O
20352555
19192158
21552609
20365774
1180281.02
Calibration curves
Images of Our PCR Negative and Positive Controls
Positive:
Negative:
PCR Results: PCR concentrations solved
Table 5: PCR Solved
PCR Product TUBE LABEL
MEAN (of RAWINTDEN DROP-BACKGROUND)
PCR Product Concentration (ug/mL)
Total Dilution
Initial PCR Product Concentration (ug/mL)
5.N
11959565.33
30.34248902
1/12
364.1098682
5.P
12861658.67
26.98004485
1/12
323.7605382
5.1-1
16922577
11.84346124
1/12
142.1215349
5.1-2
24743114
-17.30664778
1/12
-207.6797734
5.1-3
23713982.33
-13.47068354
1/12
-161.6482025
5.2-1
18118536.33
7.385666983
1/12
88.6280038
5.2-2
12791335.33
27.2421666
1/12
326.9059992
5.2-3
9405315.333
39.86314802
1/12
478.3577762
PCR Results: Summary
Our positive control PCR result was calculated to be 323.7605382 μg/mL.
Our negative control PCR result was calculated to be 364.1098682 μg/mL.
Obviously our data collection or calculations are off because we have a higher concentration in our negative control than our positive control, and they have little difference. Our calibration curve has the opposite slope of what we would expect.
Observed results
Patient 5.1 (65206): The images looked relatively normal--not much green observed, but a little. We calculated an average concentration of DNA of -75.7354803 μg/mL, which of course doesn’t make since, so our data collection and analysis could be off.
Patient 5.2 (61695): The images looked relatively normal--again, not much green observed, but a bit. We calculated an average concentration of DNA of 297.9639264 μg/mL, which is at least a positive number, but we have no proper comparison to our positive and negative controls, because our calibration curve is off, and our positive and negative concentrations were calculated to be approximately equal.
Conclusions
Our quantitative data analysis went awry. Maybe we had too much blue light interference. We are unable to conclude if either of our patients was positive for the SNP because our qualitative analysis was unclear, and our quantitative data analysis didn't work properly. We tried!
BME 100 Fall 2017
