The pipetting tutorials were very helpful for the completion of this lab exercise. Our group was able to come into the lab with an underlying knowledge of micropipetting because of the pre lab activities. Due to the pre-lab exercises we easily understood the difference between the first and second stop on the pipettor. The first stop was used before placing the pipettor into the liquid, which was then slowly released in order to bring the liquid into the pipettor. The second stop was used to deposit the liquid into our sample tubes. However, we didn't have the same amount of liquid in each of our final reactions and there were varying small amounts of leftover liquid in our tubes containing the DNA samples and PCR reaction mix. Our labeling scheme was effective and helped us to identify which PCR reactions were ours.
Fluorimeter Procedure
Smart Phone Camera Settings
Type of Smartphone: iPhone 5
Flash: off
ISO setting: 800
White Balance: N/A
Exposure: N/A
Saturation: N/A
Contrast: N/A
Camera set-up
Place the phone into the cradle vertically, make sure the phone isn't slanted, adjust the height of the fluorimeter in order to obtain the best image of the drop on the phone. The drop should be focused on the top of the image. In order to focus the image the screen of the phone can be tapped on where the focus point is.
Distance between the smart phone cradle and drop = 6 cm
Placing Samples onto the Fluorimeter
As a method of calibration of the fluorimeter, take 160 μL of water using the micropipettor and place it in the middle column of your glass slide in the shape of a drop so that the the drop covers the first two circles in that column.
Then, turn on the blue LED light and adjust if needed such that the light passes through the center of the drop.
Next, set up your camera in the cradle the necessary distance from the fluorimeter such that the camera is focused on the drop of water and is getting a nearly-perfect side view. Be sure to record this distance to ensure consistency for the rest of your lab.
Suck up the water drop with your micropipettor and discard it in your liquid waste cup.
Now, take 80 μL of the SYBR Green I solution and place it on the second two circles in the middle row of your slide.
On the same drop, add 80 μL of the first concentration of the calf thymus solution.
Adjust your LED light so that it is passing through the center of the drop.
Focus your camera and press the capture button, quickly closing the lid of the light box within that three seconds to ensure good picture quality.
Take two more images of the same sample drop to make sure your picture is focused.
Take the light box off the fluorimeter without moving the cradle with the smartphone.
Remove the drop from the slide with the micropipettor and dispose of it in the liquid waste cup.
Repeat steps 5-11 with the remaining concentrations of the calf thymus solution.
This exact same procedure will be repeated with the PCR reaction samples of your group. In order to create the solutions that will be tested, transfer 100 μL from your PCR tubes into the respective matching buffer tubes."
After you mix the buffer with your DNA, the solutions are ready to be placed on the fluorimeter and analyzed. Perform steps 5-11.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
5 μg/mL sample
.5 μg/mL sample
0 μg/mL sample
Calibrator Mean Values
Initial Concentration of 2X Calf Thymus DNA solution (micrograms/mL)
Final DNA concentration in SYBR Green I solution (µg/mL)
Our positive control PCR result was 15205.83946 μg/mL
Our negative control PCR result was 7887.674857 μg/mL
Observed results
Patient 86057 : The images of this patient were dark with very little if not any luminescence. They showed very little green light compared to the positive droplets. The observed concentration for this patient had an average of 6465.6796 μg/mL.
Patient 31866 : The images of this patient showed more green light, which was very similar to the positive droplets. Rather than a mostly black image, these images showed luminescence inside of them. The observed concentration of the droplets from this patient had an average of 11421.334 μg/mL.
Conclusions
Patient 86057 : When looking at the qualitative data, this patient appears to be similar to the negative control due to the lack of green light within the droplets. Also, the average concentration of 6465.6796 μg/mL was very close to the concentration of the negative sample which was 7887.674857 μg/mL. Due to these factors it was concluded that this patient was negative.
Patient 31866 : When looking at the qualitative data for this patient, the droplets are similar to the positive control images which contain more green light throughout the droplets. Also, the average concentration of 11421.334 μg/mL was very close to the concentration of the positive sample which was 15205.83946 μg/mL. Due to these factors it was concluded that this patient was positive.
BME 100 Fall 2015
