The pre-lab reading and interactive practice activities helped us figure out how to properly carry out the procedure for pipetting during the PCR experiment. Only one of our group members had used the micropipette before, so it was helpful to see instructions and examples for how to use it correctly. The examples also showed the common mistakes that lead to errors in data and how to avoid them.
The difference between the first and second stops were easy to recognize, and we did not have any trouble using the correct stop when drawing in the sample liquid. It made sense to use the first stop to draw up the liquid for the experiment, and then use the second stop to release the liquid, as pushing down on to the second stop clears out a larger volume in the pipette than was drawn in. Following that procedure ensures that all of the liquid was expelled from the pipette. The lab group took special care to not rush through each trial, to draw in and expel liquid slowly using resistance, and to eject the tip from the micropipette in between trials to keep them sanitary and avoid contaminating data.
The final reactions did not contain the exact same amount of liquid; visually, some of the tubes contained less liquid than others. This means that some tubes had less than 100 microliters of PCR mix after running through the thermocycler. This could have been caused by air bubbles in the pipettes that were used when creating the PCR reaction mix, or not drawing in enough liquid. These errors could potentially lead to discrepancies in our data because it alters the concentrations being used in the experiment. Even though not all of the tubes contained the same volume of PCR mix, the group ensured that all of the liquid from each tube was extracted and added to the DNA samples to mix. Then, the mixture was added to the SYBR GREEN I on the flourimeter.
We did not have to change the labeling scheme for this experiment. Everything was consistent and followed the instructions for organizing the tubes properly.
Fluorimeter Procedure
Imaging set-up
SMARTPHONE 1: Iphone 6
Placing Samples onto the Fluorimeter
Place the flourimeter on the table and turn it on.
Feel for the smooth side of the slide with your gloves on.
With the smooth side of the slide facing down, place it in the fluorimeter.
Place smartphone on cradle with the timer set up to take a photo after 3 seconds.
Adjust the fluorimeter to get a focused view of the slide. The camera should be aligned with the edge of the slide so that a side view of the droplets on the flourimeter is visible.
Using the micropipette, place 80 uL of SYBR Green I solution on the first 2 clear circles in the middle of the slide. The SYBR Green I solution should be directly in the middle and evenly dispersed across the two circles.
Place 80 uL of the sample (Calf thymus calibration liquid or PCR mix) on top of the SYBR Green I drop.
Adjust the slide so that the light illuminates the center of the drop and is refracted out of the other side in a narrow light beam.
Adjust the distance between the smartphone and the fluorimeter to have a better view. Make sure that the camera is focused again. Record the distance between the cradle and the slide.
Cover the flourimeter with the light box, but leave one flap open.
Again make sure that the drop is focused.
Press the timer button on the phone to take the picture and close the lightbox completely. Repeat this step 3 times to collect 3 images of each sample.
Using the micropipette, remove the 160 uL drop from the slide and discard into the waste liquid container. Eject the tip from the pipette and get a new one.
Move the slide to the next position, centered in the next two circles. This ensures a new, clean surface for the next sample so it is not contaminated.
Repeat steps 1-14 until you have used each of the five possible measurement positions on the slide. Then, get a new slide for the next sample and repeat until 3 images have been collected for each sample.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
1. 5 μg/mL sample
2. 0.5 μg/mL sample
3. 0 μg/mL sample
Calibrator Mean Values
Final DNA concentration in SYBR Green I solution (µg/mL)
Sample Number
RAWINTDEN
DROP -
BACKGROUND
MEAN
Standard Deviation
image 1
image 2
image 3
2.5
C-1
15934117
17052613
16673909
16553546.33
568879.3406
1
C-2
15364742
14915986
14469573
14916767
447585.011
0.5
C-3
12645681
10959209
10737067
11447319
1043738.655
0.25
C-4
13336413
10755977
13420649
12504346.33
1514717.934
0.125
C-5
5809866
5825469
5309874
5648403
293278.4961
0
C-6
11261945
7770134
7606975
8879684.667
2064710.255
Calibration curves
Images of Our PCR Positive and Negative Controls, Respectively
Our positive control PCR result was 47.7869331 μg/mL
Our negative control PCR result was -4.431475 μg/mL
Observed results
Patient 96704 : In each of the images taken of Patient 96704 (G10 1), there is no green light evident in the droplets. The green light illuminates the target DNA in each of the droplets and indicates a concentration value. The images of the drops for this patient resemble the negative control group because no green light can be seen in any of the images for any of the trials.
Patient 36904 : Similarly, in each of the images taken for Patient 36904 (G10 2), there is no trace of green light in any of the droplets. This indicates that this patient is also negative for the diseased SNP in their DNA.
Conclusions
Patient 96704 : Negative. The qualitative analysis of the images taken in this experiment indicate that there is no DNA present in the droplets. Also, the calculated initial PCR concentration for the positive control sample was approximately 47.78 ug/mL, whereas for the negative control sample the PCR concentration was -4.431 ug/mL. The concentration values calculated for Patient 96704 were much closer to that of the negative control sample than the positive control sample, averaging at approximately -16.61 ug/mL. Therefore, this patient's DNA does not contain the diseased SNP in their DNA.
Patient 36904 : Negative. The qualitative analysis of images taken for this patient also indicate that the target DNA (disease) is not present in the droplets. Also, as stated above, the calculated initial PCR concentration for the positive control sample was approximately 47.78 ug/mL, and -4.431 ug/mL for the negative control sample. The concentration values calculated for Patient 36904 were much closer to that of the negative control sample than the positive control sample, averaging at approximately -15.19 ug/mL. Therefore, the patient does not have the disease SNP in their DNA.
BME 100 Spring 2017
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