The pre-lab reading was very interactive and informative. For those who have never used a micropipette, the pre-lab information was beneficial. The first stop on the pipette is used to collect the sample that is being transferred and the second stop is used to release the sample liquid. The final reaction will have the same amount of liquid. There was no liquids left in the tubes of the DNA samples and PCR reaction mix.
We did not change our labeling scheme, but due to the tubes being very small we could not fit the whole label onto it. We labeled the first tube as G10 + and the second tube as G10-, but for the rest of the tubes we did not add "G10" to the labeling. Rather, we labeled the other 6 tubes as 1-1,1-2,1-3,2-1,2-2, and 2-3.
Fluorimeter Procedure
Imaging set-up
A member in our group provided their iPhone 6s camera in order to take images while the fluorimeter measures the fluorescence of the DNA. This is the transmittance of the wavelength given off by the DNA that is excited. The wavelength of light is exciting the DNA and if it fluoresces then there is double-stranded DNA present. Taking the iPhone 6s, the camera's flash was inactived in order to get a reasonable image of the calibration with the DNA concentration. The iPhone 6s was placed on to a metal card holder and the metal card holder was put on top of a plastic stand at a distance of 4 centimeters. The iPhone 6s was leaning on an angle due to the metal card holder and we wanted to have the camera perpendicular to the fluorimeter. In order to do this, we placed a folded piece of paper behind the iPhone 6s to prop it up.
Once our set up was completed, we began capturing images of our DNA.
A black box was placed over the fluorimeter and camera in order to ward off excess light. Before closing the box and omitting all light, the iPhone 6s timer was set at 10 seconds in order to allow the camera to focus on the new lighting exposure. The lid of the box was closed and after 10 seconds the camera took the photo. The procedure for capturing the image was repeated three times per DNA sample.
Placing Samples onto the Fluorimeter
Procedure
Calibration procedure
Calibration Procedure
1. A fluorimeter was placed on a flat hard surface adjusting the height with stacked objects to accommodate the height of the smartphone camera
2. Using gloves, one slide was placed into the fluorimeter with the smooth side down.
3. The smart phone camera app was initiated, set to an 8 sec shutter release and placed into the cradle.
4. The camera and fluorimeter were adjusted to get a horizontal edge-on view of the slide.
5. The micro-pipette was set to 85 µL to ensure the full 80 µL of solutions were added.
6. A clean pipette tip was pressed onto the micro-pipette.
7. The micro-pipette was pushed to the first position dispensing 80 µL of SYBR Green I solution onto the slide to form a beach ball shaped droplet on the first 2 circles on the slide.
8. The micro-pipette tip was discarded into the disposal cup.
9. A new tip was placed onto the micro-pipette and 80 µL of DNA thymus solution was added to the center of the SYBR Green droplet.
10. The blue light was turned on, the slide was adjusted to illuminate the center of the drop and through the other side.
11. The distance from the smartphone camera lens and the fluorimeter were adjusted to an average distance of 8cm.
12. The fluorimeter and the smartphone were covered with a light box, one side of the light box was left open the flash was turned off and the focus adjusted.
13. The camera timer was initiated and the light box flap was lowered to ensure darkness within the light box and the picture was taken.
14. After the photo was checked to ensure clarity, the 160 µL drop was removed from the slide.
15. The slide was then advanced to the next position ensuring no solution residue was present at that slide location.
16. These subsequent steps were repeated for all 5 Calf Thymus DNA solutions, taking three pictures of each for a total of 15 photos for the calibration section.
PCR Reaction Procedure
1. Obtain PCR reaction samples from instructors.
2. Each RED DOT (buffer) tube was labeled to match the 8 PCR reaction samples created in part C.
3. The micro pipette was then set tp0 100 micro liters to ensure a full draw of 100 micro liters o the PCR sample.
4. Using a new tip, the 100 micro liters of PCR sample were placed into the buffer solution tube with the duplicate label.
5. This was done for all 8 samples refreshing the tip between each transfer.
6. The caps of each tube were secured and the tube was inverted to ensure the buffer DNA mix.
7. Steps 1-16 used for the calibration samples were repeated with the SYBR GREEN I solution and the PCR reaction/Buffer solution.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA 5 μg/mL DNA sample 0.5 μg/mL DNA sample 0 μg/mL DNA sample
Calibrator Mean Values
RAWINTDEN DROP-BACKGROUND
RAWINTDEN DROP-BACKGROUND
RAWINTDEN DROP-BACKGROUND
Initial Concentration of 2X Calf Thymus DNA solution (micrograms/mL)
Final DNA concentration in SYBR Green I solution (µg/mL)
Sample Number
Image 1
Image 2
Image 3
Mean
Standard Deviation
5
2.5
C-1
62340563
56779681
28365187
49161810.33
18223762.2
2
1
C-2
31023532
31570307
32750900
31781579.67
882851.72
1
0.5
C-3
29342990
27835695
19230059
25469581.33
5455887.99
0.5
0.25
C-4
42475213
2863945
66503660
37280939.33
32136251.97
0.25
0.125
C-5
11652572
15057462
19458988
15389674
3913796.92
0
0
C-6
11600278
12714394
13923961
12746211
1162168.19
Calibration curves
Images of Our PCR Negative and Positive Controls Positive Control PCR Sample Negative Control PCR Sample
PCR Results: PCR concentrations solved
PCR Product TUBE LABEL
MEAN (of RAWINTDEN DROP - BACKGROUND)
PCR Product Concentration (µg /mL)
Total Dilution (µL)
Initial PCR Product Concentration
Positive
9431671.667
-1.056832833
0.08
-0.088069403
Negative
4796648.667
-1.520335133
0.08
-0.126694594
Patient 1-1
6975292
-1.3024708
0.08
-0.108539233
Patient 1-2
5522674.667
-1.447732533
0.08
-0.120644378
Patient 1-3
12002167.33
-0.799783267
0.08
-0.066648606
Patient 2-1
7843444.667
-1.215655533
0.08
-0.101304628
Patient 2-2
41200677.33
2.120067733
0.08
0.176672311
Patient 2-3
10835099
-0.9164901
0.08
-0.076374175
PCR Results: Summary
Our positive control PCR result was -0.088069403 μg/mL
Our negative control PCR result was -0.126694594 μg/mL
Observed results
Patient 77434 : All three images looked blurry and bright, similar to our negative control PCR sample. The initial PCR product concentration for Patient 77434 were measured as -0.108539233 μg/mL, -0.120644378 μg/mL, and -0.066648606 μg/mL.
Patient 77218 : Two of the three images looked blurry and bright, similar to our negative control PCR sample. However, the third one looked more clear like the postive control PCR sample image. The initial PCR product concentration for Patient 77218 were measured as -0.101304628 μg/mL, 0.176672311 μg/mL, and -0.076374175 μg/mL.
Conclusions
Patient 77434 : The patient's values were close to the negative PCR results, therefore, we concluded that patient 77434 was negative.
Patient 77218 : The patient's values were sporadic but closer to positive PCR results, therefore, we concluded that patient 77218 was positive. (Note: Although we said Patient 77218 was positive, The Gold Standard labeled the patient negative.)
BME 100 Fall 2016
