This lab dealt heavily with Polymerase Chain Reactions and the detection of pathogens. We use PCRs to multiply and amplify certain sequences of DNA. Since the PCR procedure is very complicated, it requires a number of supplies including Tag DNA Polymerase, a reaction buffer, template DNA and deoxyribonucleotides primers. For our lab, we were instructed to test for a pathogen in two different patients. After the template DNA, Tag Polymerase and primers were placed in a tube, they were mixed and placed into a thermocycler. This process separates the DNA and the primers duplicate the targeted segment of DNA. This entire process allows for the lab group to detect the specified pathogen in the two patients DNA.
Group 14 found that the prelab materials were very useful during this lab. We used the prelab readings as a basis to learn how to complete the micro pipetting for the replicate DNA of the two patients. A member of our group has had previous experience working in a similar lab situation, so we were able to differentiate between the first and second stop with little trouble. All of the final reactions had exactly the same amount of liquid due to careful pipetting. Additionally, at the completion of the lab there was no excess liquid in the DNA samples and PCR reaction mix. Since we took the time to label our tubes carefully and accurately during the first part of this lab, we did not have to revisit the labeling.
Fluorimeter Procedure
Imaging set-up
Our team first found a flat surface to place the camera and the box. We placed the iPhone 6 into the phone holder and leveled it using notebook paper. We staked our samples on different boxes we found to raise the samples to be leveled with the iPhone lense. The samples were raised to the correct height when we looked on the screen of the phone at eye level and saw the drop shape of the sample. When we captured our images we put the cover over so there would be a pure picture. After every picture we confirmed that each picture was focused and was not blurry.
Placing Samples onto the Fluorimeter
Using gloves, find which side of the slide is the “smooth” glass back.
Turn on fluorimeter and place it on table.
Place a slide in fluorimeter with the smooth side down.
Adjust height of fluorimeter (if needed) to get a camera view of the slide nearly edge on.
Place 80 uL drop of SYBR Green I solution on first 2 clear circles in middle of slide.
Place 80 uL drop of sample/calibration solution on top of the SYBR Green I drop.
Adjust the slide so that the light illuminates the center of the drop.
Cover with lightbox, but keep one flap up.
Make sure droplet is in focus.
As you put the flap down take picture.
Remove the drop from slide and discard liquid in waste liquid container.
Move the slide to the next position (center of next two circles).
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
(1) 5 μg/mL sample
(2) 0.5 μg/mL sample
(3) zero DNA
Calibrator Mean Values
Initial Concentration of 2X Calf Thymus DNA solution
Final DNA concentration in SYBR Green I solution (µg/mL)
Sample Number
RAWINTDEN DROP - BACKGROUND
MEAN
Standard Deviation
(µg/mL)
Image 1
Image 2
Image 3
5
2.5
C-1
8722721
8726700
841261
6096894
4551512.126
2
1
C-2
5582815
5560250
5304232
5482432.333
154737.8878
1
0.5
C-3
5473255
5469271
5382837
5441787.667
51091.6225
0.5
0.25
C-4
3792077
3858805
3594819
3748567
137266.1361
0.25
0.125
C-5
2986747
3150828
3174538
3104037.667
102266.1547
0
0
C-6
2162654
2179780
2086915
2143116.333
49419.3016
Calibration curves
Images of Our PCR Negative and Positive Controls
(1) Negative Control
(2) Positive Control
PCR Results: PCR concentrations solved
PCR Product TUBE LABEL
MEAN (of RAWINTDEN DROP - BACKGROUND)
PCR Product Concentration (µg /mL)
Total Dilution
Initial PCR Product Concentration
(Step 5 calculation)
(µg /mL)
(Step 6 calculation)
G14 +
5580913.333
487987.4186
12
5855849.023
G14 -
1730035
151271.0371
12
1815252.445
G14 1-1
1926789.333
168475.0101
12
2021700.121
G14 1-2
3874476
338778.4968
12
4065341.961
G14 1-3
1578819.333
138048.9127
12
1656586.952
G14 2-1
2346009.333
205131.1257
12
2461573.509
G14 2-2
1498387
131016.0018
12
1572192.022
G14 2-3
1252024.333
109474.333
12
1313691.996
PCR Results: Summary
Our positive control PCR result was 5855849.023 μg/mL
Our negative control PCR result was 1815252.44 μg/mL
Observed results
Patient 28550: The droplets of the first and third trials remained clear showing no green dye. However, the second trial’s droplet fluoresced a green color. The initial PCR product concentrations were 2021700.1 µg /mL, 4065342 µg /mL, 1656587 µg /mL
Patient 55154: The droplet remained clear and did not indicate any green dye. The initial PCR product concentrations were 2461573.5 µg /mL, 1572192.02 µg /mL, 1313692 µg /mL
Conclusions
Patient 28550:The patient 28550 had two droplets remain clear and one that illuminated. Since the droplets remained clear more than it illuminated, it can be concluded this patient tested negative.
Patient 55154: The patient 55154 had all its’ droplets remain clear. It can be concluded that this patient tested negative.
BME 100 Fall 2017
