For our group, it was fairly easy to pipette the samples in order to set up the reactions due to our prior knowledge and experience from previous labs. The pre-lab reading helped to give us a general understanding of what was involved in part c of the lab. We understood the difference between the first and second stop, it's clear when using the pipette which one is which. It's not easy to get all of the liquid out using the pipette, therefore some of the liquid still remained. There wasn't anything when it came to labeling scheme we really had to change, it was simple and remained the same throughout.
Fluorimeter Procedure
Imaging set-up
Our team captured the images using an iPhone. We set up the phone about four centimeters from the experiment and lined up the camera for the shot. We then placed the slide onto the fluorimeter with the smooth side facing down. We used several trays to align the iPhone camera and the fluorimeter. Our group then set up a timer for the phone to take the picture. Before we took the picture, we took the box that we were given and placed it around the experiment to keep the light from interfering with the fluorescent solution. These steps allowed our group to take the most accurate pictures that we could of the final solution.
Placing Samples onto the Fluorimeter
Using a pipette, we dropped 80μL of SYBR Green Dye onto the fluorimeter slide.
Then, we placed 80μL of DNA on top of the SYBR Green Dye.
Next, we turned on the blue LED fluorimeter light.
After that, we took the light-blocking box we were given and placed it over the sample.
We then took three pictures of the sample we had just prepared.
Finally, we removed the sample we had just photographed in order to prepare the next sample.
We then repeated all steps until all DNA solutions were photographed.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
Calibrator Mean Values
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)
G12+A
-5167.667
-19.50875773
960
-18728.40742
G12-B
57184.3333
-13.88892895
960
-13333.37197
G12 1-1 C
29738.3333
-16.36265585
960
-15708.14962
G12 1-2 D
7181.66667
-18.39570377
960
-17659.87562
G12 1-3 E
-172487
-34.58936458
960
-33205.79
G12 1-1 F
-383281
-53.58837314
960
-51444.83822
G12 1-2 G
-166368.67
-34.03791528
960
-32676.39867
G12 1-3 H
-49585.333
-23.51215259
960
-22571.66649
PCR Results: Summary
Our positive control PCR result was 18.72μg/mL
Our negative control PCR result was 13.33 μg/mL
Observed results
Patient (1) 58682 : For the most part all of the images for this one was just a liquid ball with blue, and there was one image let was sort of green, ranging from values at 15-23 for (ug/ML)
Patient(2) 54981 : A lot of these images are green, and only a couple where close to blue. these values ranged from 23 to 53 (ug/mL)
Conclusions
Patient(1) Negative : We said that this one was negative because most of the images didn't have any green in them, and then after compiling the final solutions they were more in line with the negative control's number.
Patient(2) Positive : The reason we said this one was positive was because most of the images showed up with green in them, and the numbers generally correlated with that of the positive control.
BME 100 Fall 2016
