For Part C of the lab, our group prepped our samples for the Polymerase Chain Reaction. To complete this we needed to know proper lab procedure such as how to pipette and label. The pre-lab was helpful in informing us of the steps in the process of pipetting. The first step was to label the tubes. We were able to maintain the labeling structure that we established in the initial parts of this lab. Once we had the pipette it was easy to fell the difference between the first and second stop. It takes more force to get to the second stop than it does to get to the first stop. While pipetting, it was difficult to ensure that all the liquid was all collected. It took a few tries on the first few tubes to ensure all the liquid was empties. By the end we were able to ensure that majority of the 50 microliters of the PCR mix and each sample were pipetted into the tubes. The overall process was tedious because of the amount of time it took but it was fairly simple and easy to understand.
Fluorimeter Procedure
Imaging set-up
When setting up our device to capture the images, we placed the phone, which in this case was an iPhone 7, in an upright position focused on the drop. We placed the phone approximately 7cm away from the drop and used a timer to capture the image. We used an eraser to help hold it in position while it was in the stand and then we used a folder to cover the back of the fluorimeter to keep all of the light out.
Placing Samples onto the Fluorimeter
Step 1: First, we placed 80 microliters of SYBR Green 1 and 80 microliters of a calf thymus on the slide.
Step 2: Then, we placed the box over the stand we created to meet up with the height of the camera.
Step 3: Next, we aligned the drop to meet up with the LED light.
Step 4: Then, we focused the camera on the drop.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
(1) 5 μg/mL sample
(2) 0.5 μg/mL sample
(3) zero DNA
Calibrator Mean Values
Initial Concentration of 2X Calf Thymus DNA solution (μg/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
0
0
C-6
14278351
13427774
14185926
13964017
466693.702
0.25
0.125
C-5
19226506
17073922
46566069
27622165.67
16441168.13
0.5
0.25
C-4
15721858
48006477
46168607
36632314
18132286.65
1
0.5
C-3
23466266
24932157
225536611
91311678
116244512.5
2
1
C-2
19196572
17899556
21911378
19669168.67
2047239.519
5
2.5
C-1
21911378
21225181
21391583
21509380.67
357943.8481
Calibration curves
Images of Our PCR Negative and Positive Controls
(1) Negative control PCR sample
(2) the Positive control PCR sample
PCR Results: PCR concentrations solved
PCR Product TUBE LABEL
MEAN (of RAWINTDEN DROP - BACKGROUND)
PCR Product Concentration (µg /mL)
(Step 5 calculation)
Total Dilution
Initial PCR Product Concentration (µg /mL)
(Step 6 calculation)
G16P
17325033.67
-2.112494389
12
-25.34993267
G16N
9674297.333
-3.387617111
12
-40.65140533
G161-1
11230618.75
-3.128230208
12
-37.5387625
G161-2
25825943.33
-0.695676111
12
-8.348113333
G161-3
12141144
-2.976476
12
-35.717712
G162-1
15417670
-2.430388333
12
-29.16466
G162-2
16374828.67
-2.270861889
12
-27.25034267
G162-3
10571880.33
-3.238019944
12
-38.85623933
PCR Results: Summary
Our positive control PCR result was -25.34993267 μg/mL
Our negative control PCR result was -40.65140533 μg/mL
Observed results
Patient 30857 : This patient appeared to be negative because the sample was clear, just like the negative control.
Patient 75331 : This patient appeared to be positive because the sample was fluorescent green, just like the positive control.
Conclusions
Patient 30857 :Patients initial concentration values were closest to positive control. So, the patients results are positive.
Patient 75331: Patients intial concentration values were closest to negative control. So, the patients results are negative.
BME 100 Fall 2016
