We collected the materials from the front of the classroom and were able to identify the DNA samples of the two different patients by their patient ID numbers. We had minor difficulties finding the positive and negative control solutions, but we were ultimately able to collect and identify all the materials with relative ease. We all have had experience in micropipetting; we rotated the micropipetting tasks between all members and had one team member hold open the lids of the tubes while another team member micropipetted the solutions. We experienced no difficulties executing the task and there were no spills or lost samples. We did not encounter any issues with placing the tubes into the thermal cycler. We also double-checked all of our tube identities and temperatures for the different thermal cycling stages before initiating the thermal cycling process.
The pre-lab activities definitely helped us to understand today's lab procedure and pipetting techniques. The video on micropipetting was especially helpful to us and after practicing a few times with the two different stops, we felt adequately confident about our micropipetting techniques. We understood that the first stop was used to draw up the liquid, and the second stop was used to eject the liquid. The final reactions did have exactly the same amount of liquid, because of the accuracy of the micropipettor. There was a marginal amount of liquid remaining in the tubes that contained the DNA samples and the PCR reaction mix, but this should not significantly affect the results of the experiment. We did not have change our labelling scheme.
Fluorimeter Procedure
Imaging set-up
We collected a light box, four glass slides, a metal mesh stand for the smartphone, and the box cover to shield from light. In order for the sample/SYBR GREEN liquid on the glass slide to be in view of our smartphone, we placed 3 plastic cartons under the light box to boost the box's height. We adjusted the phone on a phone stand and ensured that the flash was turned off. Once the phone was positioned, we did not move it to ensure consistent images. To take the photograph, we set the timer on the camera application of the phone to 3 seconds, hit the capture button and quickly closed the flap of the box cover, and waited until the photograph was taken.
Placing Samples onto the Fluorimeter
Set the micropipettor volume to 80 microliters.
Depress the micropipettor plunger to the first stop.
Holding the plunger to the first stop, carefully place the micropipettor tip in the SYBR GREEN I tube.
Release the plunger. Wait a few seconds and then remove the micropipettor from the tube.
Position the micropipettor over the middle of the three glass "windows" or "dots" in the second row on the glass slide. Carefully depress the plunger to the second stop.
Eject the used tip into a waste beaker.
Repeat the same micropipetting process for the DNA sample. Place the DNA sample onto the same dot as the SYBR GREEN I, so that the two liquid "bubbles" form a larger bubble of liquid.
Using this micropipetting technique for placing samples upon the fluorimeter:
Place 80 microliters of SYBR GREEN I onto a glass "dot" on the glass slide.
Add 80 microliters of the DNA sample (calf thymus or patient DNA) to the existing SYBR GREEN I drop.
Turn on the light (switch located on the side of the box) and align the drop with the light source so that the drop is illuminated.
Take 3 pictures using the aforementioned device setup: Set the timer on the phone to 3 seconds. Hit the capture button and quickly close the flap on the light box to ensure no stray light gets through when the picture is taken. Repeat this step for each picture.
Using the micropipettor, draw up the SYBR GREEN I and DNA on the slide using. Depress the plunger to the first stop, place the tip in the fluid, and release the plunger. Eject the tip still full of liquid into the waste beaker. Repeat as many times as necessary to remove all fluid on the slide.
Move the slide to align a new row of glass dots with the light source, ensuring that the new sample will not be contaminated by fluid from the previous run.
Repeat steps 1-6 for all samples: calf thymus DNA 5, 2, 1, 0.5, 0.25; patients G1-1, 1-2, 1-3, 2-1, 2-2, 2-3.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
High Calf Thymus DNA:
Low Calf Thymus DNA:
Zero Calf Thymus:
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
MEAN
Standard Deviation
Image 1
Image 2
Image 3
5
2.5
C-1
12126631
15591926
16371472
14696676.33
2259595.706
2
1
C-2
25638821
30633405
27921041
28064422.33
2500377.169
1
0.5
C-3
29985680
30827502
30178782
30330654.7
440981.9158
0.5
0.25
C-4
29816494
33867835
34304690
32663006.3
2474810.078
0.25
0.125
C-5
13609318
12955818
13526375
13363837
355780.1478
0
0
C-6
17555970
17493087
17603265
17550774
55272.47717
Calibration curves
Images of Our PCR Negative and Positive Controls
Positive Control:
Negative 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 (µg /mL)
Positive
17574994
17574990.59
12
210899887.1
Negative
8269022.333
8269018.921
12
99228227.05
G8 1-1
83127195
83127191.59
12
997526299.1
G8 1-2
15896780.67
15896777.25
12
190761327.1
G8 1-3
7789431
7789427.588
12
93473131.05
G8 2-1
12183142
12183138.59
12
146197663.1
G8 2-2
29013196.67
29013193.25
12
348158319.1
G8 2-3
12213523
12213519.59
12
146562235.1
PCR Results: Summary
Our positive control PCR result was 210,899,887.1 μg/mL
Our negative control PCR result was 99,228,227.05 μg/mL
Observed results
Patient 21105 : The droplet is clear with a small circular area inside that appears darker. There are no visible signs of fluorescent green, only the blue color produced by the light box. The average PCR result of the three samples of Patient 1 is 427,253,585.7 μg/mL.
Patient 12210 : Similar to Patient 1, the droplet is clear with a small circular area inside that appears darker. There are no visible signs of fluorescent green, only the blue color produced by the light box. The average PCR result of the three samples of Patient 2 is 213,639,405.7 μg/mL.
Conclusions
Patient 21105 : The PCR results of Patient 1 are much higher than the PCR results for the positive control, so one can assume that the patient has positive DNA results.
Patient 12210 :The PCR results of Patient 2 are very close to the PCR results for the positive control, so the patient has positive DNA results.
BME 100 Fall 2018
