The Pre-Lab reading insured that the individual mircopipetting knew exactly what they were doing to the letter and not trying to remember anything from experience they had several years ago. Depressing the plunger to the first stop, placing it in the solution, and letting the plunger go, allowed for the desired amount of fluid to be sucked into the tip. Depressing the plunger to the second stop allowed for the fluid to be completely ejected from the tip. While all the correct measurements were taken with the micropipettor and all the fluid was ejected from the tip, there appeared to be an extremely small amount of fluid left in the original sample tubes of patient DNA. Knowing this it is impossible to say that there was the same amount of fluid in each of the final reactions. The labeling scheme stayed constant throughout the entire process.
Fluorimeter Procedure
Imaging set-up
The fluorimeter was turned on and the slide was placed in such a way that the beam of fluorescent light was directly between two of the printed circles. The camera was then placed 1.5 inches away on a phone stand. The phone was set to take 3 pictures in 5 second intervals. The distance allowed for the slide to be moved towards the phone without a change in distance, relative to the phone and drop. The cover was then placed over the entire set up. The phone was focused on the drop in the darkness. The pictures were then set to be taken and the cover was completely closed. 17 seconds were allowed to pass before we removed the cover.
Placing Samples onto the Fluorimeter
Step one: the slide was placed on the Fluorimeter such that the beam of light passed between the first 2 circles.
Step two: 80 microliters of CYBR Green dye was then placed between the two circles with a fresh micropipette tip, after the tip is disposed.
Step three: 80 microliters of diluted PCR solution are placed on top of the previous drop with a fresh micropipette tip, after the tip is disposed.
Step four: A cover is placed over the set up completely enveloping it in darkness.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA Calibrator Mean Values
Initial Concentation of 2X Calf Thymus DNA solution (micrograms per liter)
MEAN
5
19697954.33
2
18730361
1
20138941.67
0.5
24384070.67
0.25
13819524.33
0
-1051196
Calibration curves
Images of Our PCR Negative and Positive Controls
PCR Results: PCR concentrations solved
PCR Product TUBE LABEL
MEAN( of RAWINTDEN DROP - BACKGROUND)
PCR Product Concentration (ug / mL)
Total Dilution
Initial PCR Product Concentration (ug/ML)
1-1
11990496
0.3317493333
12
3.980992
1-2
6437871.333
0.5936881111
12
7.124257333
1-3
5872323.667
0.6879460556
12
8.255352667
2-1
4919932.333
0.8466779444
12
10.16013533
2-2
5437013.667
0.7604977222
12
9.125972667
2-3
4625081.333
0.8958197778
12
10.74983733
PCR Results: Summary
Our positive control PCR result was 0.95756 μg/mL
Our negative control PCR result was -0.85911 μg/mL
Observed results
Patient 40178 : While the Pictures had no green inside of them, the Image J gave higher values to the patient 40178 pictures in general then Patient 92225, and the PCR product concentrations did not help clarify this.
Patient 92225 : The pictures for Patient 92225 did show hints of green, but the actual results seem to differ with the values from image j.
Conclusions
Patient 40178: If a conclusion were to be made, it would be that patient 40178 was negative, as the values of this patient were closer to that of the negative PCR Product concentration.
Patient 92225: If a conclusion were to be made, it would be that the patients 40178 was postive, due to the visible amount of green and the closer ug/mL values to the Postive test.
BME 100 Fall 2017
