The pre-lab helped us to correctly pippete our samples. Without it we could've possibly mishandled and cross contaminated our samples. The final reactions didn't have the same amount of liquid. There was trace amounts of liquid left in the tubes, but not a significant amount. We didn't have to change our labeling scheme.
Fluorimeter Procedure
Web camera set-up
Take the camera stand and place it next to fluorometer. Put phone on stand and set it up to take pictures of sample so that the picture is clear and close up.
Placing Samples onto the Fluorimeter
Step 1: Place necessary amount of SYBR green onto middle of first two dot rows.
Step 2: Place neccesary amount of DNA sample into the SYBR green dot
Step 3: Align the slide so the fluorescent light shines into the optic piece on other side
Step 4: Put cover box over system.
Step 5: Set camera timer to 3 seconds and take picture, making sure to use the cover box to block out light
Step 6: Take 3 pictures for each sample.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
High
Low
Zero
Calibrator Mean Values
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 DRO-BACKGROUND
Mean
Standard Deviation
5
2.5
C-1
2186129
2188483
2191151
2188587.667
2512.635535
2
1
C-2
2056335
2042466
2040840
2046547
8515.555002
1
.5
C-3
2087348
2088135
2102797
209276
8701.199285
.5
.25
C-4
1643382
1627741
1625240
1632121
9832.158512
.25
.125
C-5
1420245
1441205
1449159
1436869.667
14936.57207
0
0
C-6
767545
808998
716664
764402.3333
46247.15293
Calibration curves
Images of Our PCR Negative and Positive Controls
Positive
Negative
PCR Results: PCR concentrations solved
PCR concentrations solved
PCR Product Tube Label
MEAN (of RAWINTDEN DROP - BACKGROUND)
"PCR Product Concentration (µg /mL)
(Step 5 calculation)"
Total Dilution
"Initial PCR Product Concentration
(µg /mL)
(Step 6 calculation)"
G7+
8154383.333
12.94575944
0.08335
1.079029049
G7-
5398040.667
7.958194833
0.08335
0.6633155393
G7 1-1
5527930.333
8.193228419
0.08335
0.6829055887
G7 1-2
3753018.667
4.981549874
0.08335
0.415212182
G7 1-3
5557640.333
8.246988261
0.08335
0.6873864715
G7 2-1
7128031.667
11.08859004
0.08335
0.92423398
G7 2-2
8313322
13.2333568
0.08335
1.103000289
G7 2-3
7451784.333
11.67441609
0.08335
0.9730625814
PCR Results: Summary
Our positive control PCR result was 12.946 μg/mL
Our negative control PCR result was 7.958 μg/mL
Observed results
Patient 56256 : The images from patient 56256 looked like the negative control, and had a PCR concentration of ~ 8 microliters.
Patient 98031 : The images from patient 98031 looked like the positive control, and had a PCR concentration of ~ 12 microliters.
Conclusions
Patient 56256 : Patient 56256 appeared to test negative, from analysis of data.
Patient 98031 : Patient 98031 appeared to test positive, from analysis of data.