At the beginning of the lab, each member of the team took turns micro-pipetting 50 micro-liters of PCR reaction mix into PCR tubes. Only one member had prior experience with micro-pipetting, so they assisted the other group members pipette the samples. The pre-lab resources were very helpful to the team in order to understand concepts with micro-pipetting and comprehend the PCR reaction procedure. In the micro-pipetting procedure, The first stop on the pipette was understood as the extraction mechanism, used to absorb samples when released in a solution. The second stop was understood as the release mechanism, used to eject the samples completely into the tubes.
When the PCR reaction was completed successfully, a small amount of liquid remained in the origional tubes and was placed into the biohazzard wastebag. The tubes that fit in the DNA cloning machine were filled with 50 microliters of the PCR reactin mix. The team had received prior warning to labeling these containers, so each tube was labeled on its side where the letters would be best read and preserved.
Fluorimeter Procedure
Imaging set-up
In order to set the camera to capture the images of the flourimiter procedure, the lens of the camera was placed horizontally level to the test slide. To do this, the mechanism holding the slide was raised with leveling equipment to achieve an ideal height level with the camera lens. The camera was stood upright to avoid errors in image capture. On the slide that was placed in the laser mechanism, droplets of mixed SYBR green and DNA mix solution were placed between the circular divots, holding the droplets still for analysis. Each test was performed between a different set of dots as to not cross-contaminate the samples. The camera and laser mechanism were placed into a dark container as to best capture the fluorescent colors produced by the reaction. The droplets appeared translucent until a laser shone through the samples where it revealed a greed-blue hue from the center of each applicable drop. From this, the students were able to determine the amount of DNA present in each sample.
Placing Samples onto the Fluorimeter
Use a micropipettor to transfer 160 microliters of water to the slide. The drop should be placed in the center of the first two rows.
First, make sure the smartphone is placed on the fluorimeter so that a clear image of the side of the bubble is shown, make sure the phone is atleast 4 cm away to ensure the image is not blurry.
Use a ruler to find the distance between the drop and the smartphone and record this value
Align the slide with the blue light so that it is focused on the center of the droplet
Set the camera to delay 3 seconds before taking a picture and then depress the camera button and close the light box to get the least amount of light in the picture
Take 2 more images following same process
Remove the 160 microliters of water from the slide
Add 80 microliters of SYBR GREEN I and 80 microliters of the calf thymus samples
Repeat previous process until there is a total of 18 images
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
5 μg/mL
0.5 μg/mL
0 μg/mL
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
19920
22425
20609
20984.66
1294.06
2
1
C-2
19558
21258
19520
20112
992.64
1
0.5
C-3
10791
10603
12997
114.63
1331.22
0.5
0.25
C-4
10047
9755
9223
9675
417.78
0.25
0.125
C-5
9201
9102
7508
8603.66
950.16
0
0
C-6
7774
9021
7848
8214.33
699.57
Calibration curves
Calibration Curve with all Values
Calibration Curve without value for 5μg/mL sample
Images of Our PCR Negative and Positive Controls
Positive Control PCR Sample Negative Control PCR Sample
Our positive control PCR result was 35.312777001 μg/mL
Our negative control PCR result was 4.144401468 μg/mL
Observed results
Patient 77020 : The images of patient 77020 were shaded almost black, which is similar to the negative control picture. Thus displaying Patient 77020 is closer to the negative control than the positive control. When calculating the PCR results of the DNA concentration, Patient 77020 had an average concentration range near the negative PCR result of 4.144401468, indicating Patient 77020 displayed a negative PCR result.
Patient 86836 : When comparing the images of Patient 86836 to the control pictures, Patient 86836 had an image shaded quite dark as well. Indicating that Patient 86836 resulted in a negative PCR result. Patient 86836 had an overall concentration closer to the negative PCR result of 4.144401468. Thus proving the overall PCR result for Patient 86836 is negative.
Conclusions
Patient 77020 : Patient 77020 PCR resulted negative for the DNA sequence. The negative control did not glow green because there was no DNA in its droplet, which was similar in the results of our own average sampling. The DNA was therefore not contained in the patient sample.
Patient 86836 : Patient 86836 also resulted negative. When comparing the images of the positive control and the negative control to Patient 86836's images, Patient 86836's images more closely resembled the negative control pictures over the positive control pictures. Patient 86836 had an overall concentration of DNA close to the negative control of 4.144401468, thus proving that Patient 86836 does not contain the disease.
BME 100 Fall 2018
