Initially going into this lab, none of our team members knew the proper procedures of micro-pipetting and this is where the pre-lab videos and reading came into use. We all watched the pre-lab demonstration video explaining step-by-step how to properly use a micro-pipette and the proper implications of the first and second stops on the pipettor. The first stop was used when ejecting the liquid from the tube and into the pipettor, and the second stop was used when inserting that liquid into another container. We all practiced the technique and designated the person that was best at micro-pipetting to be the user of the device for the remainder of the lab. We ran into a couple of times in which not all of the liquid was extracted from the initial tube, but we remedied this issue by inserting the liquid back into the same tube and repeating the process again being a little bit more careful the second time.
When we initially received the primer tubes from our lab TA, they each had exactly 51 micro-liters of liquid inside. After we had micro-pipetted all of the samples, most of the samples had a little bit of liquid (approximately 1 micro-liter) while others were completely empty. This is most likely due to the fact that we had mistakenly not extracted all of the liquid on the first try and had to re-do the sample a couple of times. This repeat most likely caused us to accidentally extract all of the primer instead of leaving that 1 micro-liter behind, giving us an error of about 2 micro-liters in the final reaction.
Finally, our labeling scheme stayed the same throughout the lab because after extracting the liquid from the initial containers, they were empty and could be put to the side with no further use while the new containers contained the DNA and the primer. Therefore we could use the same labeling scheme without having to worry about getting the tubes mixed up.
Fluorimeter Procedure
Imaging set-up
For this lab, we had a stand called a fluorimeter that was built to hold glass slides, and in the middle of the stand was a laser light that we aimed at our droplets to measure the fluorescence that each drop had. To take the pictures, we placed the stand in a black box to minimize the amount of light exposure the droplet received and then set the camera of our iPhone 7 Plus inside the box with no flash and maximum exposure and saturation to the get the clearest picture possible of each droplet.
Placing Samples onto the Fluorimeter
Set the fluorimeter up and turn it on.
Put the glass slide into the grooves of the fluorimeter with the smooth side of the slide facing down
Put the phone in the stand so that the camera is level with the glass slide.
Using the micropipettor, add an 80 microliter drop of the SYBR Green solution on the first two dots of the glass slide.
Place an 80 microliter drop of the sample solution into the drop of SYBR Green.
Adjust the slide so that the light is going through the droplet.
Slide the black box over the fluorimeter while using the flap to focus on the drop with the phone camera.
Start the camera timer while it is focused on the droplet and put the flap down to eliminate external light exposure.
After the picture is taken and checked to make sure it is good quality, use the micropipettor set at 160 microliters to suck the droplet up and discard it in the liquid waste cup.
Move the glass slide up so the next set of dots is in the light and repeat steps 4-9, doing three trials for each solution.
Once the glass slide runs out of space, discard it into the sharps container and place a new one onto the fluorimeter (see step 2).
Once complete, discard all liquid waste and samples into the designated bin and give any remaining SYBR Green solution to the lab instructor.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
0.0 μg/mL Sample of Calf Thymus (Zero)
0.5 μg/mL Sample of Calf Thymus (Low)
5 μg/mL Sample of Calf Thymus (High)
Calibrator Mean Values
Initial Concentration of 2X Calf Thymus DNA solution (µg/mL)
Final Concentration DNA concentration in SYBR Green I solution (µg/mL)
Patient 10452 : From the images taken of Patient 10452, there was no sign of any green fluorescence in the droplet, closely resembling the negative control. The similarities in these two droplets gave the indication that there was little to no disease markers in this patient. All of the trials were consistent in shape, size, and color confirming the initial indication. To quantitatively confirm our findings, we calculated the concentration of green color in each of the images. The calculated concentrations from Patient 10452 were -5.80 μg/mL, -8.17 μg/mL, and -5.09 μg/mL. This coupled with our qualitative findings confirmed that there were no disease markers in Patient 10452.
Patient 45121 : From the images taken of Patient 45121, there was no sign of any green fluorescence in the droplet, closely resembling the negative control. The similarities in these two droplets gave the indication that there was little to no disease markers in this patient. All of the trials were consistent in shape, size, and color confirming the initial indication. To quantitatively confirm our findings, we calculated the concentration of green color in each of the images. The calculated concentrations from Patient 45121 were -3.39 μg/mL, -4.28 μg/mL, and -4.79 μg/mL. This coupled with our qualitative findings confirmed that there were no disease markers in Patient 45121.
Conclusions
Patient 10452 : The calculated concentrations from Patient 45121 were -3.39 μg/mL, -4.28 μg/mL, and -4.79 μg/mL. These values were most similar to those of the negative control which is what was expected based on our qualitative findings. This is because there was no sign of green fluorescence in any of the droplet samples. Therefore, Patient 10452 does not have any indication of the disease marker.
Patient 45121 : The calculated concentrations from Patient 45121 were -3.39 μg/mL, -4.28 μg/mL, and -4.79 μg/mL. These values were most similar to those of the negative control which is what was expected based on our qualitative findings. This is because there was no sign of green fluorescence in any of the droplet samples. Therefore, Patient 45121 does not have any indication of the disease marker.
BME 100 Spring 2018
