Pipetting the samples to set up the PCR reaction of this lab was relatively simple and easy after going through the pre-lab content. The pre-lab content was comprehensive in use of the pipettes and were very helpful with how to properly pipette the samples. The first and second stop on the pipette serves different purposes- the first stop is for use when measuring a specific amount of fluid, the second stop is used when expelling the liquid from the pipette in order to provide extra pressure to ensure all liquid is expelled. The final reactions all contained the same amount of liquid as they should, and there was no liquid remaining in the DNA samples tubes. Our labeling scheme was consistent with what we had predetermined.
Fluorimeter Procedure
Imaging set-up
To set up the imaging process for the fluorimeter procedure, we stacked a few containers on top of one another to get a desirable height to take pictures of the slides. We also used a stand to hold an iPhone 7 in place. For the camera, we turned off the flash, and set ISO to 800 to get the best picture that we possibly could of each slide. We ensured that the camera was at least four centimeters away from the drop on the slide allowing the picture to be the best that it could be.
Placing Samples onto the Fluorimeter
While wearing proper PPE (gloves/lab coat), identify which side of the slide is the smooth glass back.
Place the fluorimeter on the table and turn it on.
Place the smooth side of the slide downwards and slide it into the fluorimeter.
Open the camera app on the smartphone, set the camera timer for three seconds, and place the phone on the cradle, four centimeters away from the fluorimeter.
Adjust the height of the fluorimeter so that the camera view of the slide is looking straight on.
Using the micropipette, place 80 microliters of the SYBER green solution on the slide.
Obtain a new tip for the pipette and measure 80 microliters of the sample solution and pipette it to the top of the Cyber Green I drop.
Adjust the slide so that the light is pointed directly in the center of the drop and the drop projects the light on the other side.
Place the lightbox over the fluorimeter and the smartphone and keeping one side flap up.
Ensure that the drop is focused on the camera.
Set the smartphone to take a picture in three seconds, and close the flap before the picture is taken.
Once the picture is taken, remove all 160 microliters from the slide and dispose the liquid in a biohazard waste container.
Move the slide up so that the light is pointing to the next two circles on the slide.
Repeat steps 1-14 until all five possible measurement positions of the slide are used.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
5 μg/mL sample
0.5 μg/mL sample
0 μg/mL sample
Calibrator Mean Values
Initial Concentration of 2X Calf Thymus DNA solution (μg/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
12424542
12645326
12574607
12548158.33
112743.2598
2
1
C-2
28620247
26942819
28681574
28081546.67
986643.6917
1
0.5
C-3
19869589
21544429
21666382
21026800
1004027.444
0.5
0.25
C-4
12104715
12488353
12691996
12428354.67
298202.269
0.25
0.125
C-5
13877645
13511632
13193636
13527637.67
342285.2817
0
0
C-6
3824427
3989135
4041076
3951546
113110.11
Calibration curves
Images of Our PCR Negative and Positive Controls
Negative Control Sample
Positive Control Sample
PCR Results: PCR concentrations solved
PCR Product Tube Label
Mean
PCR Product Concentration (μg/mL)
Total Dilution
Inital PCR Product Concentration (μg/mL)
G6 +
14651073.67
0.665107367
12
7.9812884
G6 -
11054826.33
0.305482633
12
3.6657916
G6 1-1
11087711
0.3087711
12
3.7052532
G6 1-2
16918800.67
0.891880067
12
10.7025608
G6 1-3
11139325.33
0.313932533
12
3.7671904
G6 2-1
10983041.33
0.298304133
12
3.5796496
G6 2-2
8826582.667
0.082658267
12
0.9918992
G6 2-3
7940007.333
-0.005999267
12
-0.0719912
PCR Results: Summary
Our positive control PCR result was 7.9812884 μg/mL
Our negative control PCR result was 3.6657916 μg/mL
Observed results
Patient 10548 : The images for this patient showed some green, but it looks mostly blue from the light. The results were 3.71, 10.70, and 3.77 μg/mL.
Patient 96514 : The images for this patient showed absolutely no green, only blue for all three images of Patient 96514. The results were 3.58, 0.992, and -0.0720 μg/mL.
Conclusions
Patient 10548 : The qualitative results suggest that this patient is positive, with the green coloring. But the quantitative results suggest that the patient was more negative, since two out of the three results were closer to the negative control.
Patient 96514 : The qualitative results suggest that this patient is negative, due to the lack of green coloring. The quantitative results suggest the patient is most likely negative, as the numbers are all closer to the negative control. But two out of the three results are much smaller, with even a negative result, which suggests an inconclusive result.
Gel Electrophoresis
Lane Labels
1: DNA Ladder
2: Positive Control
3: Negative Control
4: Patient 1, replicate 1
5: Patient 1, replicate 2
6: Patient 1, replicate 3
7: Patient 2, replicate 1
8: Patient 2, replicate 2
9: Patient 2, replicate 3
The results of the gel electrophoresis are somewhat hard to see in this picture, however they were more clear in person. The first lane had the DNA Ladder, which is pretty visible in the image. The second lane has the positive control, which shows a mark close to where the DNA Ladder ends, indicating a positive result. The negative control in lane three shows no mark, as is should, indicating a negative result. The next three lanes, lanes 4-6, all also contain marks at the same spot as the positive control. This is an indicator that all three replicates of the first patient returned a positive result, which is good considering it is consistent. For the three DNA replicates for the second patient in lanes 7-9, there is a haziness that makes it difficult to see in the image. However, in person, there appeared to be no mark, indicating a consistent negative result for patient 2. These results are consistent with the qualitative results obtained in the Fluorimeter testing.
BME 100 Spring 2017
