For this lab, our experience with pipetting the samples to set up the reaction was overall successful. The pre-lab reading was helpful in that it explained proper pipetting technique. This enabled us to work efficiently. The difference between the first and second stop of the pipettor was straightforward to understand. We pressed on the top of the pipettor gently until it reached a slight barrier, which was the first stop. Pressing all the way so that the top was fully pushed in was the second stop. During the pipetting, there were a few instances where the pipette didn't extract the full 50 μL from the PCR reaction mixture. In this case, we simply dispensed the amount initially taken, then used a new pipette tip to take out leftover mixture. We used two strips of four test tubes, with the first containing test tubes G7 +, G7 -, G7 1-1, G7 1-2 and the second containing G7 1-3, G7 2-1, G7 2-2, G7 2-3. Also, we did not have to change our labelling scheme. Finally, we placed our PCR test tubes into a thermal cycler that was shared with Group 6.
Fluorimeter Procedure
Imaging set-up
To set up our device to capture images from the fluorimeter, we first used two pipette boxes to lift the fluorometer up to the height of the Samsung phone's camera. Simultaneously, we set up ImageJ on our computer to later collect the image data. We placed the phone in the cradle, then measured and moved it approximately 5 centimeters away from the fluorimeter. We positioned the phone facing the side of the fluorimeter where the droplet would be closest to the camera. Once the phone was positioned, we set a timer for 5 seconds on the phone and ensured that it would take three images in quick succession. When photos needed to be taken, the camera would be started and the lightbox would be placed over the fluorimeter for about 5-10 seconds. We took care to move the phone as little as possible throughout the experiment.
Placing Samples onto the Fluorimeter
Slide the sample slide into the slot at the top of the Fluorimeter, smooth glass side down with the tab in the front
Turn on the fluorimeter
Position the slide so that the green light is located in between the first two rows of circles
Take a micropipette, set it to 80ml, and attach a clean tip
With the micropipette measure and place a droplet of SYBR Green in between the row of circles lit by the green light
Remove the tip into the waste bin and replace with a new one
With the micropipette measure and place 80ml of the sample DNA onto the SYBR Green droplet
Set the timer on the camera phone and place black box over the fluorimeter
Allow camera time to take three pictures
Remove black box cover
Place new tip on the micropipette
Using the micropipette, suck up the droplet of SYBR Green and sample DNA
Place the used tip and liquids into the waste bin
Repeat steps 4-9 for each set of sample DNA
Once all of the rows in a slide have been used, discard in sharps container
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
zero DNA in ImageJ
0.5 μg/mL sample in ImageJ
5 μg/mL sample in ImageJ
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 Image 1
RAWINTDEN DROP - BACKGROUND Image 2
RAWINTDEN DROP - BACKGROUND Image 3
MEAN
Standard Deviation
5
2.5
C-1
14259666
14248369
14223226
14243754
18653.26718
2
1
C-2
13740609
13736453
13775852
13750971
21647.25766
1
0.5
C-3
12222525
12232085
12211842
12222151
10126.6903
0.5
0.25
C-4
11711536
11757941
11713376
11727618
26276.88734
0.25
0.125
C-5
8599344
8679099
8692615
8657019
50403.40882
0
0
C-6
3116446
3390462
3460011
3322306
181640.0933
Calibration curves
Calf Thymus DNA Calibration Curve 1
Calf Thymus DNA Calibration Curve 2
Images of Our PCR Negative and Positive Controls
Positive control PCR sample in ImageJ
Negative control PCR sample in ImageJ
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)
G7 +
9579887
0.64497175
12
7.739661
G7 -
1302030
-1.4244925
12
-17.09391
G7 1-1
9865592.7
0.716398175
12
8.5967781
G7 1-2
9887714.3
0.721928575
12
8.6631429
G7 1-3
9710386.3
0.677596575
12
8.1311589
G7 2-1
571865
-1.60703375
12
-19.284405
G7 2-2
1146509.7
-1.463372575
12
-17.5604709
G7 2-3
985475.33
-1.503631168
12
-18.04357401
PCR Results: Summary
Our positive control PCR result was 7.74 μg/mL
Our negative control PCR result was -17.09 μg/mL
Observed results
Patient 51980 : The images taken in the fluorimeter for this patient showed a bright green color within the droplet of SYBR Green I and sample. Quantitatively, the replicates of initial PCR product concentration were 8.60 μg/mL, 8.66 μg/mL, and 8.13 μg/mL.
Patient 45751 : The images taken in the fluorimeter for this patient showed no bright green color within the droplet of SYBR Green I and sample. The droplet was essentially a clear blue. Quantitatively, the replicates of initial PCR product concentration were -19.28 μg/mL, -17.56 μg/mL, and -18.04 μg/mL.
Conclusions
Patient 51980 : These patient's results were all within less than 1 μg/mL of the positive control value. Based on the experimental values, it can be concluded that this patient is most likely positive for the disease SNP.
Patient 45751 : These patient's results were all within about 2 μg/mL higher or lower than the positive control value. Based on the experimental values, it can be concluded that this patient is most likely negative for the disease SNP.
BME 100 Fall 2017
