The technique of micro-pipetting used in experiment C was used for transferring the sample DNA from the patients to the buffer containing the elements for the PCR reaction. The tutorial for this lab was significantly helpful, especially since the interactive tutorial simulated the laboratory experience. This activity helped recognize the difference between the first and second stop of the pipette. The first stop was used when extracting a certain amount of fluid from the sample and the second stop was used to release the liquid into another container. This activity also highlighted the importance of following directions in the correct order of steps since doing so lessens the chance of error.
The final reaction did not have the same amount of liquid. There was approximately a 20% standard deviation from the theoretical 100 microliters. This was due to experimental errors such as air bubbles and inexperience using this technique. Such factors must be considered when analyzing the results as possible sources of error and discrepancies. This was because there was a change in the concentrations of the solutions tested.
However, when transferring the PCR reactions to the buffer in section four of experiment D, the micropipette was set to 100 microliters so equal amounts of the solution were transferred for the test. Some liquid in the tubes remained since there was an excess of solution from the PCR reactions. The labeling scheme was not changed and was consistent during all the experiment for all the procedures (including the DNA Electrophoresis), as stated in the lab manual.
Fluorimeter Procedure
Imaging set-up Smartphone: Iphone 7
Placing Samples onto the Fluorimeter
Turn on the fluorimeter by using the switch on the side, a blue LED light should indicate that it is on.
While wearing gloves, take a slide and carefully find its smooth side.
Place the slide with the smooth surface facing up on top of the fluorimeter.
Take the the smartphone and open the camera app. Before taking any picture remove the its flash settings and then set its timer to 3 seconds.
Place the smartphone on the cradle and reposition the fluorimeter to where the flat surface of the slide can be clearly captured by the camera lense. Record the distance between the cradle and the fluorimeter.
Using a micropipette take 80 uL of SYBR Green I Solution and place a drop of it on the first two circles of the middle column of the slide.
Now add another drop of 80 uL of Sample/Calibration Solution on top of the of the SYBR Green I drop.
Ensure that the blue LED light is hitting directly on the drop and can be reflected to the other side of the fluorimeter.
Place the light box over both the flourimeter and the cradle with the front flap open.
Check the camera for focus one last time then start the camera timer. Close the flap and allow time for the camera to take the pictures before opening it again.
Using a micropipette remove the 160 uL drop from the slide and place the liquid and the tip used in a waste liquid container.
Adjust the slide so that the LED light now hits the next two circles in the middle column.
Continue to repeat steps 6 through 12 until you have taken images of 5 different positions in the slide.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA 5 μg/mL sample
0.5 μg/mL sample
zero DNA
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
885684
906951
828859
873831.3333
40372.6937
2
1
C-2
1089590
1039356
995380
1041442
47139.62847
1
0.5
C-3
355639
350930
304423
336997.3333
28308.28614
0.5
0.25
C-4
358210
473649
463558
431805.6667
63935.11308
0.25
0.125
C-5
312854
265933
277372
285386.3333
24465.63333
0
0
C-6
209162
221406
253404
227990.6667
22844.19001
Calibration curves
Images of Our PCR Negative and Positive Controls Negative Control Sample
Our positive control PCR result was 9.688298086 μg/mL
Our negative control PCR result was -0.3219983088 μg/mL
Observed results
Patient 84453 : The picture of Patient 84453 was observed to have a dark blue color throughout all of the pictures. There was observed to be slight brightness or glow in the pictures. It was observed to have a concentration 0 μg/mL.
Patient 40501 : The picture of Patient 40501 was observed to have a light blue color and seemed to glow more than Patient 8453. The pictures led to an observation that the concentration was .5 μg/mL.
Conclusions
Patient 84453 : Since this patient's Initial PCR Product Concentration for all three replicates were more similar to the value of the negative control rather than the positive control, it was concluded that the patient was negative.
Patient 40501 : Similar to Patient 84453, Patient 40501's initial concentration for all three replicates were more similar to the negative control concentration. Therefore, it was also concluded that this patient was negative.
DNA Electrophoresis (Extra Credit)
Image I: DNA Electrophoresis Results
Column 1: Ladder for comparison
Column 2: Positive Control
Column 3: Negative Control
Column 4-6: Patient 1 (samples 1.1, 1.2, 1.3)
Column 7-9: Patient 2 (samples 2.1, 2.2, 2.3)
Conclusion:
DNA Electrophoresis technique leverages from the negative polar charge of the DNA molecule and separates it into fragments as it is moved to a positive charge, from an anode to a cathode. The ladder placed in the first column of the agarose gel serves as the comparison for the fragments of all the other samples. In this experiment, the positive and negative controls were placed in the second and third columns respectively. Notice how, qualitatively, a fragment of in the positive control can be distinguished near the bottom of the ladder. The negative control does not exhibit any fragment as it is expected.
In this experiment, each of the three samples from patient one and two results in negative results. This suggests that either this patient results are negative or that an error occurred during the procedure of the experiment. Comparing these results with the ones obtained using the fluorimeter test, the evidence suggests that this is more likely due to the nonpresence of the altered SNPs in the patients that an experimental error in the procedure. Therefore, we conclude the negative result for both patients one and two, in each of their samples.
BME 100 Spring 2017
