Our group found that watching the pre-lab videos, and reading all the material assisted greatly when it came the time to actually conduct the experiment. At first, we did not understand the difference between the first and second stop on the pipettor, but after reviewing the pre-lab material once more it became easier and more well understood. All final reactions did end with the same amount of liquid (100 microliters) due to careful pipetting. Once the pipetting was completed, there was no liquid left in either the DNA sample tubes or in the tube containing the PCR reaction mix. The labeling scheme that was suggested in the workbook worked very well for our group and helped us keep the tubes differentiated from each other and easily identifiable.
Fluorimeter Procedure
Imaging set-up
All the necessary materials were provided in the lab. We had a fluorimeter to make the DNA glow, a dark box to block light so that the florescence could be observed better , glass slides to test our samples on, and an iPhone 6 camera to capture the image. The glass slide was set up on the fluorimeter and the PCR solution and SYBR green dye were mixed in the center of the glass with a micropipette (80 microliters each drop). When this was complete, we put the box over the fluorimeter and turned the device on. We had to make adjustments accordingly to make sure that the camera was lined up properly to take a good photograph of the drop. The iPhone 6 camera was then eye level with the glass slide and images were captured. Sometimes the images did not turn out perfectly and the image had to be retaken, but the drop stayed intact so it was not difficult to redo, only small adjustments to the placement of the phone so that it would have a better focus. After the image was captured and agreed to be a proper picture, the drop was picked up with the pipettor and this was repeated for each necessary substance.
Placing Samples onto the Fluorimeter
Place a drop of SYBR Green I solution in-between the first two middle circles on the slide.
Place a drop of solution (the same amount as SYBR Green I) on top of the SYBR Green I solution drop
Adjust the slide for the light to illuminate the center of the drop.
Place smartphone on the cradle with the camera app open and with the timer set to 3 seconds.
Adjust height of fluorimeter to get a camera view of the slide at eye level (edge on).
Adjust the distance between the smartphone and the fluorimeter to view the drop closely but keeping it in focus (> 4cm away) and record the distance.
Cover with the light box, keep one flap open and then make sure that the drop is still focused.
Set the 3 second camera timer and then close the last flap of the light box.
Remove all liquid from the slide and discard in the appropriate liquid waste container.
Adjust the slide for the light to illuminate the center of the next two circles.
Repeat all steps above until they have been completed for all possible measurement positions on the slide.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
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
2323300
2451357
2336582
2370413
70413.43077
2
1
C-2
826856
8360347
8189145
8272685.333
85675.37711
1
0.5
C-3
5773862
5797464
5579813
5717046.333
119432.0076
0.5
0.25
C-4
13141781
13259096
13181363
13194080
59682.44192
0.25
0.125
C-5
8082285
8051025
7964259
8032523
61149.62937
0.0
0.0
C-6
4839431
5044754
4772866
4885683.667
141722.4506
Calibration curves
Images of Our PCR Negative and Positive Controls
PCR Results: PCR concentrations solved
PCR Results: Summary
Our positive control PCR result was 0.2167345555 μg/mL.
Our negative control PCR result was 0.1754024445 μg/mL.
Observed results
Patient 34668: For this patient, we used 80 μg of both PCR reaction mix and SYBR green, so the drop was a total of 160 μg. For this patient, the G2 1-1 sample appeared very illuminated in the image, and was a light blue color. The G2 1-2 sample appeared half dark blue on the bottom, and half of the light blue (similar to the first sample) on the top half of the drop. The G2 1-3 sample image appeared completely a dark blue color with little to no illumination.
Patient 59805: For this patient, we used 80 μg of both PCR reaction mix and SYBR green, so the drop was a total of 160 μg. For this patient, the G2 2-1 sample, and the G2 2-2 sample appeared the same in the image. They were half illuminated on the top of the drop (a light blue color), and a dark blue color on the bottom half of the drop. The G2 2-3 sample appeared almost completely illuminated in the image with the exception of a small part of the bottom of the drop which was not illuminated, so it appeared as a dark blue color.
Conclusions
Patient 34668: This patient compares more to the positive control because the positive control was very illuminated when the experiment was completed. It appeared a very light blue color in the image which is the same appearance that the G2 1-1 image portrayed. On the contrary, this patient is not very similar to the negative control because the image of it was illuminated on the bottom half but not illuminated on the top half. We drew the conclusion that this patient had a successful PCR reaction and produced a lot of DNA because the drop was very illuminated by the SYBR Green.
Patient 59805: This patient compares to the negative control because the negative control was not illuminated in the image when the experiment was completed. It appeared a dark blue color in the top of the image and a light blue color on the bottom of the image which is the same appearance that the G2 2-1 and G2 2-2 portrayed in their images. On the contrary, this patient is not very similar to the positive control because the image of it was completely illuminated. We drew the conclusion that this patient didn't have as successful of a PCR reaction as the other patient because the drop in the fluorimeter was not completely illuminated, so there was less DNA in the drop.
BME 100 Fall 2017
