Name: Anwar Gadhi Roles: Mutant Rat Sensei and Adoptive Father. He's also pretty lit.
Name: Jayashree Iyer Role: Fiery, Violent Bad Boy
Name: Ivan Quidera Roles: Comedian and Party Dude
Name: Joshua Lugay Rodil Role: Brave Leader
Name: Katie Wilkinson Roles: Scientist, Inventor, Engineer, and Technological Genius
LAB 5 WRITE-UP GROUP 16
PCR Reaction Report
Preparing the PCR samples for the thermocycler involved micropipetting PCR reaction mix into multiple DNA samples. There was a positive control, negative control, and three replicates each for two patients. The PCR reaction mix included the "taq" polymerase, primers, and dNTPs (deoxyribonucleotide triphosphates). 50 μL of the PCR reaction mix was pipetted into 50 μL of each DNA sample. The pre-lab reading helped when pipetting the reaction mix out of the tube: the micropipettor should only go to the first stop, and when pipetting that reaction mix into the DNA sample, the micropipettor should go to the second stop to ensure all excess liquid is expelled from the pipette tip. All the final reactions in the eight PCR tubes had the same amount of DNA sample/reaction mix, 100 μL, because we had slowly pipetted to reduce the incidence of air bubbles. Each tube was labeled with our group name and the type of DNA sample, then our eight PCR tubes were placed into the thermocycler with another group's eight PCR tubes to fill up the sixteen slots so that the thermocycler was completely filled up and could run the PCR cycles.
Fluorimeter Procedure
Overall, there were quite a few steps to make sure that the procedure flowed as smoothly as possible. The yield was satisfactory, in the case of our group.
Imaging set-up
We set up the black box with the fluorimeter inside and set it so that it is sheltered from overhead light, but at the same time close enough to the camera so that images could be taken.
The hydrophobic side of glass slides slid into a designated area of the fluorimeter; this is the key location of the experiment.
Our group choose the most advanced camera among our group in order to take the best possible pictures with the cleanest resolution. In our case, an iPhone X was used.
It is expected to use the following settings to make sure the camera's photo is rendered as perfect as possible: setting ISO to 800, setting white balance to auto, set exposure and saturation to the highest possible setting, while leaving contrast on the lowest possible one.
After that, several settings were modified including turning off the flash so that the SYBR Green I will not be "bleached."
Once all the settings were properly configured, # The camera is prepared so that it is about 7 centimeters away from the first 2 rows of the glass slide and at a 90 degree angle so that the patient DNA/calf thymus DNA and SYBR Green I may be visible and captured. Turn on the blue LED light switch at the side of the fluorimeter.
Camera and Fluorimeter Setup
Placing Samples onto the Fluorimeter
Firstly, we attained and prepared all the materials that are required for the procedure. This includes our positive and negative control along with patient DNA samples, SYBR Green I solution wrapped in foil because of its light sensitivity, double stranded calf thymus DNA of various concentrations (5, 2, 1, 0.5, 0.25, 0 μg/mL), the black box/fluorimeter, and glass slides that have both a smooth and hydrophobic side.
The calf thymus DNA will first be analyzed to generate a calibration curve. 80 μL of the calf thymus DNA, starting with the highest concentration (5 μg/mL) is pipetted onto the middle of the first 2 rows of the glass slide so that the drop lines up with the LED light
With a new pipette tip, 80 μL of SYBR Green I is then pipetted onto the calf thymus DNA, making the final volume of the drop 160 μL.
Check one final time that the drop is aligned in the middle of the first 2 rows of the glass slide and that the blue LED light is shining through the center of the drop. Once those standards are met, take 3 images using the camera, being careful not to move the camera or the set up.
Carefully lift the black box off of the fluorimeter. Using a new pipette tip, remove the 160 μL of solution off the glass slide, slide the glass slide out of the fluorimeter, and then insert a new glass slide.
Repeat steps 2-5 with the rest of the concentrations of calf thymus DNA (2, 1, 0.5, 0.25, 0 μg/mL) and then the 8 PCR DNA samples (positive control, negative control, 3 replicates of patient 1, 3 replicates of patient 2).
Pipetting various DNA samples and SYBR Green I onto glass slide in fluorimeter
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
Concentration of 5 μg/μL of double stranded calf thymus DNA combined with SYBR Green I
Concentration of 0.5 μg/μL of double stranded calf thymus DNA combined with SYBR Green I
Concentration of 0 μg/μL of double stranded calf thymus DNA combined with SYBR Green I
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 DROP - BACKGROUND Image 1
RAWINTDEN DROP - BACKGROUND Image 2
RAWINTDEN DROP - BACKGROUND Image 3
MEAN
Standard Deviation
5
2.5
C-1
1237738
995990
1197196
1143641.333
129466.6004
2
1
C-2
894383
882103
805558
860681.3333
48131.44511
1
0.5
C-3
600678
610273
687290
632747
47478.62828
0.5
0.25
C-4
446091
530027
722125
566081
141504.8073
0.25
0.125
C-5
142834
116453
407303
222196.6667
160848.5476
0
0
C-6
152132
261106
66252
159830
97654.82442
Calibration curves
Initial Calf Thymus DNA Concentrations Including Highest Initial Concentration
Initial Calf Thymus DNA Concentrations Without Highest Initial Concentration
Saturation is the point at which something (in this case SYBR Green I) is absorbed or dissolved in the DNA. SYBR Green I saturates at high DNA concentrations, meaning it is absorbed into the DNA and would not be shown as brightly on camera, which would affect the line of best fit. Dot Plot 2's calibration curve would be more accurate, as the highest DNA concentration is removed.
Images of Our PCR Negative and Positive Controls
PCR positive control for patient DNA samples combined with SYBR Green I
PCR negative control for patient DNA samples combined with SYBR Green I
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 (µg /mL)
G16 positive
254958.3333
0.435298945
12
5.223587343
G16 negative
357032.6667
1.066767298
12
12.80120758
G16 1-1
357876
0.562894638
12
6.754735659
G16 1-2
275583.6667
0.562894638
12
6.754735659
G16 1-3
191670.6667
0.043778793
12
0.525345511
G16 2-1
279825
0.589133044
12
7.069596526
G16 2-2
155343
0.180957153
12
2.171485839
G16 2-3
366607
1.12599755
12
13.5119706
PCR Results: Summary
Our positive control PCR result was 5.223587343 μg/mL.
Our negative control PCR result was 12.80120758 μg/mL.
Observed results
Patient 89675 (1): The initial PCR product concentrations found were 6.754735659 µg/mL, 6.754735659 µg/mL, and 0.5253455106 µg/mL.
Observed Patient 89675
Patient 76880 (2): The initial PCR product concentrations found were 7.069596526 µg/mL, 2.171485839 µg/mL, and 13.5119706 µg/mL.
Observed Patient 76880
Conclusions
Patient 89675: The disease DNA concentration in the positive control was 5.223587343 µg/mL. The 1-1 1-2 replicates, like the positive control and the negative control contains a positive value that is over 5. Meanwhile replicate 1-3 was under 1 µg/mL but also positive. However, the initial concentration of patient 1's replicate DNA is closest to the positive control, 5.2236 µg/mL. Therefore, patient 1 is most likely positive for the disease SNP (single nucleotide polymorphism).
Patient 76880: The disease DNA concentration in the negative control was 12.80120758 µg/mL. The 2-1 2-2 and 2-3 replicates, like the positive and negative controls contained positive concentrations ranging from 2 µg/mL through 13.5 µg/mL. However, the initial concentration of patient 2's replicate DNA is closest to the negative control, 12.8012 µg/mL. Therefore, patient 2 is most likely negative for the disease SNP.
BME 100 Fall 2018
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