Our group was able to successfully use the micro-pipettes to carry out the PCR reaction. Almost every member of our group had used a micro-pipette before, so their incorporation into the lab did not prove to be an especially difficult challenge. Despite having background knowledge and experience, the pre-lab reading and simulation helped us review the concepts and steps behind using a micro-pipette, and thus the pre-lab was very useful. We correctly used the pipette throughout the experiment, pressing the plunger down to the first stop when taking up the liquid and pressing the plunger down to the second stop when releasing it. In our experiment, every one of our final reactions looked like they had exactly the same amount of liquid (as expected). It is possible that there was a very slight discrepancy in size between some of the reactions, perhaps due to small bubbles, but we were careful to be as consistent with all of the liquid sample sizes as possible. There was no easily visible liquid left at the bottom of the tubes containing DNA/PCR reaction mix, however there is still a possibility that there was a very small amount remaining due to human error. We did not change our labeling scheme; it was kept consistent with that of Lab 4.
Fluorimeter Procedure
Imaging set-up
The primary objective of this section of the lab is to use fluorescence to detect DNA using a smartphone. Settings on the camera were changed in addition to the download of the computer software Image J. The materials collected were a tray of sample tubes, a micropipette, pipette tips, our patient PCR reaction tubes, glass slides, and a Fluorimeter kit, which included a fluorimeter, phone stand, and black box.
Placing Samples onto the Fluorimeter
Place the slide into the fluorimeter with the rough side facing down
Place smartphone on its stand and adjust the fluorimeter and stand until a close and in focus picture of the droplet can be taken
Pipette 80 µL of SYBR GREEN 1 in between the two rows on the slide
Pipette 80 µL of one of the Calf Thymus or PCR solutions onto the SYBR GREEN 1 drop on the slide
Align the drop and LED light by moving the slide on which the drop rests
Place box over the fluorimeter, with one flap open
Make sure the camera is still focused on the drop, then start the camera timer and close the flap
After the picture is taken, remove the box and remove the 160 µL drop using the micropipette
Discard the liquid waste into the hazardous waste container
Repeat these steps, but change where on the slide the calf thymus solution or PCR is placed (until 5 possible measurement positions are used) ensuring that both the PCR and Calf Thymus samples have been imaged in all of the possible positions
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
Image J for 5μg/mL sample:
Image J for 0.5μg/mL sample:
Image J for zero DNA sample:
Calibrator Mean Values
Initial Concentration of 2X Calf Thymus DNA solution (micrograms/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
5
2.5
C-1
4185234
5365929
6015000
5188721
927665.3007
2
1
C-2
3743365
3580604
3746131
3690033.33
94778.6734
1
0.5
C-3
2766824
2549043
2487094
2600987
146921.246
0.5
0.25
C-4
1009030
986727
872285
956014
73363.922
0.25
0.125
C-5
779044
830556
859594
823064.667
40794.187
0
0
C-6
175705
166114
563156
301658.333
226514.3904
Calibration curves
Images of Our PCR Negative and Positive Controls
Positive Control - Image J:
Negative Control - Image J:
PCR Results: PCR concentrations solved
PCR Product TUBE LABEL
MEAN (of RAWINTDEN DROP - BACKGROUND)
PCR Product Concentration (µg /mL)
Total Dilution
Initial PCR Product Concentration (µg /mL)
G4 +
4224095.7
3.236
12
38.832
G4 -
-438702
-0.8554
12
-10.2648
G4 1-1
9446639
7.817
12
93.804
G4 1-2
9341033
7.725
12
92.7
G4 1-3
8437615.3
6.932
12
83.184
G4 2-1
-384931
-0.807
12
-9.684
G4 2-2
-334290.33
-0.762
12
-9.144
G4 2-3
-1553485
-1.831
12
-21.972
PCR Results: Summary
Our positive control PCR result was 38.832 μg/mL
Our negative control PCR result was -10.26 μg/mL
Observed results
Patient 38270 : The photos from patient 38270 were observed to have a more green appearance, and when observed in Image J in the green channel, they are very light. Additionally, the quantitative results for this patient were 93.804 μg/mL, 92.7 μg/mL, and 83.184 μg/mL.
Patient 31977 : The photos from patient 31977 were observed to not omit any fluorescence under the fluorimeter, and when observed in Image J in the green channel, the images are very dark. The quantitative results for this patient were -9.684 μg/mL, -9.144 μg/mL, and -21.972 μg/mL.
Conclusions
Patient 38270 : The positive control result was 38.832 μg/mL, which was comparable to the results of Patient 38270. Therefore, it can be concluded that Patient 38270 is positive for the Disease SNP.
Patient 31977 : The negative control result was -10.26 μg/mL, which clearly parallels to the results observed from Patient 31977. Therefore, it can be concluded that Patient 31977 is negative for the Disease SNP.
BME 100 Spring 2018
