Reaction Summary
Our team worked together to preform this experiment with the most accuracy and precision possible. Before the lab, everyone in the group completed the pre-lab material, including watching the videos and completing the virtual lab activity. The videos served very well to show us the proper technique for pipetting, from the very first step to the last. The videos began with proper handling of the materials, and continued through the attachment of the tip, the withdrawal of the liquid from the first container, and then the distribution of the liquid into the second container. The main takeaway from the videos was the distinction of the first stop and the second stop on the micropipette. If the experimenter goes to the wrong stop, the experiment can be ruined. The virtual lab served to show us the purpose of these techniques, and gave us an application to relate the new information to.
While completing our experiments, we followed proper procedure very well. We had minimal error in differentiating between the first and second stop on the micropipette, and completed the combination of the samples fairly well. It did seem that all of the tubes had the same amount of liquid when they were transferred, and it didn't seem that there was marginal error in this. However, it is important to learn from this that it would be very hard to fix an experimental error in this case. Since the liquid used in this experiment is clear, if we had forgot any liquid in the tubes after combining them, we might not notice this error unless we looked very closely. This creates some room for error further than just the misuse of the pipette itself, making the pre-lab materials and proper practice even more prominent. Thankfully we avoided error, used the same labeling scheme, and preformed a decent experiment.
Fluorimeter Procedure
Imaging set-up
We gathered all the materials needed including glass slides, lightbox, stand for the smartphone(iPhone). We adjusted our phone with the stand 4cm away from the sample which was placed on a glass slide under the lightbox with the cover to make sure that the sample did not get any light from outside. We set the timer on camera at 10 seconds to make sure we get clear pictures.
Placing Samples onto the Fluorimeter
Fluorimeter slides were obtained (Note: handle slides at all times with gloved hands only).
The smooth side was identified (glass coated) on first slide and then it was placed in fluorimeter with the glass side facing down.
The camera was set-up per above, with the height adjusted as necessary to obtain edge-on view.
Utilizing a micropipette, 80uL of SYBR Green I solution was placed on the first pair of clear circles on the slide, starting in the middle row, nearest to camera. Placement resulted in a single, cohesive drop (Note: proper pipette protocol was followed at all times, with pipette tips being changed between samples to prevent contamination).
Utilizing a micropipette, 80uL of Sample/Calibration solution was placed on top of the SYBR Green I drop. Solution remained a single cohesive drop.
The fluorimeter light beam from was verified to strike the center of drop.
A 4cm distance was measured form the fluorimeter to locate proper phone placement. Phone was placed accurately.
The lightbox was then placed over the fluorimeter and phone stand while leaving the side flap open.
Drop was visualized through phone and a focused, edge-on view obtained.
The timer was initiated and the flap closed.
After the elapsed time (10sec), the flap was raised and image accuracy was verified.
Utilizing a micropipette, the 160uL drop was removed and discarded in the liquid waste container.
The slide was then prepared for the next sample by adjusting the slide position so that the next pair of circles in the center row were centered on the fluorimeter light beam.
The above steps were repeated for all calibration and patient samples as directed by the workbook.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
High-5mcgLow-0.5mcgZero-0mcg
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
5
2.5
C-1
9477729
7819480
1658249
6318486
4120170.398
2
1
C-2
11289575
11220399
11673267
11394414
243958.4306
1
0.5
C-3
9970626
9621280
8986280
9526062
499033.1778
0.5
0.25
C-4
6867496
6824316
6531089
6740967
183037.4532
0.25
0.125
C-5
10119487
9281028
10035668
9812061
461793.7218
0
0
C-6
4532101
5042459
4723820
4766127
257795.8739
Calibration Curves
Images of Our PCR Negative and Positive Controls
Negative ControlPositive Control
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)
G14 +
9948075.33
1.29905024
12
15.58860288
G14 -
10291999
1.34396085
12
16.1275302
G14 1-1
8311933.67
1.113033
12
13.1796672
G14 1-2
8423390.33
1.113033
12
13.356396
G14 1-3
11764224.3
1.53620855
12
18.4345026
G14 2-1
11045448.7
1.44234862
12
17.30818344
G14 2-2
8608675
1.12414723
12
13.48976676
G14 2-3
12158640
1.58771256
12
19.05255072
PCR Results: Summary
Our positive control PCR result was 15.6 μg/mL
Our negative control PCR result was 16.1 μg/mL
Observed results
Patient 62160: Within the images, the patient showed that he may have been within the spectrum of being positive, yet unfortunately our data was compromised causing the results when calculated to appear inconclusive.
Patient 19010: Much like the previous patient, the patient's DNA when photographed showed signs of being positive, yet when the results of the information given were calculated within the graph the results showed to be inconclusive.
Conclusions
Patient 62160: Within the results for this patient the values were rather close to the values of our positive control, however, the values were rather skewed due to the data being compromised causing the final results to be inconclusive.
Patient 19010: This patient's values were extremely close to the positive control's values, yet the information was compromised causing the results to be inconclusive.