Our team had an overall easy time with pipetting the samples. One of our lab members ran the actual pipetting and she had previous experience from her biology class last semester. The process was a bit stressful however, due to the fact that this process is very precise and requires a lot of finesse and concentration. The pre-lab reading absolutely helped with this process. It helped those in the group who already had this knowledge to refresh their memory on how to complete PCR. Those who had no prior experience with PCR were able to help as well due to the pre-lab reading. Our group does understand the difference between the first and second stop on the pipettor. When you are collecting material, you have to go up to the first stop, and when you are releasing the material, you go to the second stop. There was a very small amount of liquid left in the bottom of the tubes, so not all of the reactions had exactly the same amount of liquid. This is an example of experimental error because ideally, there would be absolutely no liquid left in the tubes. We do not have to change our labeling scheme because of this due to the fact that it was such a small amount of liquid left in the tube.
Fluorimeter Procedure
Web camera set-up
The smartphone used for this experiment was an iPhone. The camera on the phone was set up to the optimal conditions for taking pictures of the DNA in the fluorimeter.
The flash was turned off and the rest of the settings (ISO, white balance, exposure, saturation, and contrast) could not be changed.
The camera was then set on a three second timer and placed in a cradle about 5 centimeters away from the drop on the slide.
The camera was level with the drop so that the pictures of the drop would be from a completely horizontal viewpoint and could be analyzed in ImageJ.
Placing Samples onto the Fluorimeter
Place a fresh slide with the porous side facing up and center the light shining across the center of the row
Using a micropippettor, place 80 microliters of SYBR green in the middle of the first two rows of the slide so that the light is shining through the center of the drop, try to make the drop have the shape of a ball
Add 80 microliters of the PCR mix the the drop of SYBR green already on the slide
Take a few photos of the focused drop and then remove the drop of sample by using the pipettor
Move the slide over to a fresh new row and repeat steps 2-4
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
5 μg/mL sample
0.5 μg/mL sample
zero DNA
Raw Calibration Data
Calibrator Mean Values
Calibration curves
Images of Our PCR Negative and Positive Controls
PCR Negative Control
PCR Positive Control
PCR Results: PCR concentrations solved
PCR Product
RAWINTDEN DROP - BACKGROUND
PCR Product Concentration (µg/mL)
Total Dilution
Initial PCR Product Concentration (µg/mL)
G10 +
261947
1.568894033
12
18.82672839
G10 -
510055
0.02326170415
12
0.2791404498
G10 A-1
455842
3.076036223
12
36.91243468
G10 A-2
479978
2.157985347
12
25.89582416
G10 A-3
693906
2.059847895
12
24.71817474
G10 B-1
457773
0.3150394577
12
3.780473492
G10 B-2
422613
0.733495319
12
8.801943827
G10 B-3
568147
0.258862304
12
3.106347648
PCR Results: Summary
Our positive control PCR result was 1.58 μg/mL
Our negative control PCR result was 0.023 μg/mL
Observed results
Patient 99518: The image had a small fluorescent green tint of the sample while the light shined through it. There was a concentration of 1.56 μg/mL.
Patient 27737: There was not an obvious amount of fluorescence in the sample and there was a concentration of 0.26 μg/mL that was observed from the sample.
Conclusions
Patient 99518: There is a DNA concentration of 1.57 μg/mL for this patient and when compared to both the positive controls can be seen to be more closely related to the positive control which has a concentration of 1.58 μg/mL.
Patient 27737: There is a DNA concentration of 0.26 μg/mL, when compared to the control variables it was closer to the negative control, having a concentration of 0.023 μg/mL.
BME 100 Spring 2016
