Pipetting the samples ton set of the reaction was fairly simple and straightforeward. If we conducted this lab without watching the pre-lab videos of how to properly use a micropipette this lab would have had much more error occur within our PCR reaction and we would have struggled with taking photos of the cyber-green substance mixed with our samples and the different concentrations of DNA samples that we also tested. The difference between the first stop versus the second stop on the pipette is also clear with the first stop being used to accurately collect the exact amount of fluid we needed and using the second stop to then ensure that all collected fluid is expelled out of the micro-pipette tip. Using those stops integrated on the pipette ensured that all of our final reactions received the same amount of fluid, reducing any possible errors due to concentration in our data. However there was lequid left in all of our DNA tubes after we conducted the experiment using cyber-green but this is expected as we used the fluid that was in those tubes to dilute the DNA from our PCR reactions. We also did not have to change our labeling scheme because we used simple labels that were similar to the ones given on the instructions for the lab to prevent confusion within out group. As our skills improved using the pippetes within this lab, it made analyzing the effects of the PCR reaction much easier to understand as we minimized error in our data to interpret.
Fluorimeter Procedure
Imaging set-up
First we placed the call phone with a camera on (Samsung Galaxy s7 edge) it into the given stand that came in the light box. To secure the cell phone into the stand that it is in we then also placed a pencil to that the cellphone was standing almost perpendicular to the desk that it was placed on. After setting up the cell phone in its stand we then went about setting up the florimiter so that the viewing platform of it was within almost level with the camera. To do this the florimeter had to be placed onto a few of the generic trays until an optimal height to where the camera is still a few centimeters above the viewing platform of the florometer. THen collect a few glass treys that have small holes on them(provided in lab) and slide it onto the black platform with the smooth side facing down. Once all of this is complete and the Florimeter has sufficient stability, turn on the blue LED light and keep it on for the remainder of the experiment.
Placing Samples onto the Fluorimeter
First, set the micro-pipette to collect only 80μL of fluid and lock the fluid amount into place. Then firmly press the micro-pipette onto a disposable plastic tip used for drawing liquid.
Press down onto top button of the micro-pipette until you reach the first stop. Then place only the tip of the pipette into the cuber-green solution and released the button on the micro-pipette to draw in 80μL of the fluid.
Position the tip of the micro-pipette over the first two central circles closest to you on the fluorimeter stand and press the circular button on the micro-pipette all the way down until you cannot press the button down anymore and all liquid has been expelled onto the area between the two central circles closest to the camera. After all liquid is onto the tray dispose of the micro-pipette tip.
Repeat steps 1-3 but instead of the cyber-green use the 0 concentration of Calf Thymus DNA solution(water) and then take three photographs of the bubble of solution setting on the florimeter trey. When putting the next bubble of solution onto the glass trey make sure that it is 2 sets of circles on the glass trey from the last solution. Multiple glass trey panels will be needed to avoid cross contamination. Ensure that the entire florimeter is completely covered when the photo is taken by using a photo timer on the cell phone.
Dispose of all liquids on they glass trey using the micro-pipette into waste cup, use the procedure described in steps 1 and 2 to use the micro-pipette.
Repeat steps 1-4 for all concentrations of Calf Thymus DNA solution and the PCR reacted DNA samples that were ran in a previous lab. (Lab C)
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
High Calf Thymus DNA
Low Calf Thymus DNA
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 (micrograms/mL)
Sample Number
RAWINTDEN drop - background
Mean
Standard Deviation
Image 1
Image 2
Image 3
5
2.5
C-1
5818898
5794366
5840860
5818041
23258.84
2
1
C-2
5747027
5753099
5746619
5748915
3629.188
1
0.5
C-3
5647584
5694635
5696257
5679492
27645.04
0.5
0.25
C-4
5452299
5526287
5375103
5451230
75597.67
0.25
0.125
C-5
4633995
4846652
4355053
4611900
246543.2
0
0
C-6
143161
239418
161575
181385
51094.71
Calibration curves
Dot Plot 1
Dot Plot 2
Dot Plot 3
A third Dot Plot with one more data point was created to account for the high degree of saturation
Images of Our PCR Negative and Positive Controls
Positive Control
Negative Control
PCR Results: PCR concentrations solved
PCR Product Tube Label
MEAN (of RAWINTDEN drop - background)
PCR Product Concentration (micrograms/mL)
Total Dilution
Initial PCR Product Concentration (micrograms/mL)
G4 P
5919283
0.9162363
12
10.99484
G4 N
1779867
-0.02926747
12
-0.3512097
G4 1-1
2025629
0.02686820
12
0.3224185
G4 1-2
2212967
0.06965890
12
0.8359068
G4 1-3
2240593
0.07596924
12
0.9116309
G4 2-1
2367487
0.1049536
12
1.259443
G4 2-2
2137018
0.05231118
12
0.6277341
G4 2-3
2247776
0.07760987
12
0.9313184
PCR Results: Summary
Our positive control PCR result was 10.99484 μg/mL
Our negative control PCR result was -0.3512097 μg/mL (This error of a negative concentration is due to the background of the image withing the analyzed image J being more than the green within the actual droplet)
Observed results
Patient 54887 : This patient's droplet was nearly completely clear, with only a small amount of green (the same as the negative control). Also, the initial concentration in μg/mL was determined to be 0.6899854 (average of the three).
Patient 26612 : This patient's droplet was also nearly completely clear, with only a small amount of green (the same as the negative control). The initial concentration in μg/mL was determined to be 0.9394984 (average of the three).
Conclusions
Patient 54887 : Since the droplet had nearly no difference in green color when compared to the negative control, and since the initial concentration was determined to be nearly zero, the patient was determined to be negative for the DNA tested.
Patient 26612 : Also, for the second patient, there were no observable differences in green color when compared to the negative control. The value of initial concentration was nearly zero for this patient as well. Therefore this patient was determined to be negative for the DNA tested.
BME 100 Fall 2016
