When our group was pipetting the samples, we had some trouble with using it because we didn't know which button sucked and dropped then we figured out it was the same button. Also calibrating it to have the right amount of substance in the pipette was a trouble because we could not understand how to change the the amount in it.The second part of the lab saw us adding the PCR to a gel that displayed a green color when mixed and then we took multiple pictures of each PCR sample and compared each one. The ones with the most color in it had the highest amount of DNA in it and the lighter it appeared the less amount of DNA was present in the sample. The Pre-lab did help, because it gave us an insight into the lab being done and which substances were being used.This pre lab also helped us because it basically showed the lab that was being done and is also gave some key information on how to use the tubes, PCR, the DNA, and the gel. The first stop on the pipettor sucked and dropped the liquid into the tube while the second one dropped the tube inside the pipettor. The final reactions had about the same amount of liquid in it because there was 50 micro liters in the first part then another 50 micro liters was added to the sample. There was some of the sample left on the side of the tube that was unable to be taken out because the pipettor only took a certain amount of the sample so not all of it was taken out of the tube. Our labeling system was the same so there would not be any confusion about which tube was which and which tubes were ours when we gave it to the TA it ended up helping us as we were able to get our samples. Using the values taken by the experiment we were able to fill out the tables and the graphs given accordingly.
Fluorimeter Procedure
Imaging set-up
In order to capture the images a device called a fluorimeter was used. The set up of the device included a stand for the phone, a light box with a flap, two and a half PCR tube racks, the fluorimeter itself, and iPhone 7 Plus. To set up the fluorimeter, first, stack the two and a half PCR tube racks on top of each other to adjust the height of the flourimeter so that it is in line with the camera lenses. Next, place the fluorimeter on it. Then, set the stand for the phone so that the phone sits roughly 4 cm from the flourimeter, if the phone is unsteady in the stand add paper in order to fill some empty space. Finally, set the phone on a delay so that there is time to cover the set up and close the flap before a picture is taken and place the phone in the stand.
Placing Samples onto the Fluorimeter
[First the Calf Thymus and SYBR Green I solutions were prepared.]
[A pipettor with a fresh tip was used to extract 80 microliters of the of the SYBR solution from the bottle. ]
[The drop of the SYBR solution was carefully placed between two of the middle clear dots on the slide, and the tip was discarded.]
[A new tip on the pipettor was used to extract 80 microliters of the 5 micrograms/mL Calf Thymus solution]
[This drop was placed directly on top of the SYBR drop]
[The drop was then photographed with the phone set-up 3 times]
[The fluorimeter was then shifted to the next row, and the droplet was discarded using the pipettor]
[This process was repeated for each of the Calf Thymus solutions]
[Once each of the PCR reaction samples were combined with 100 microliters of buffer using a pipettor, the process explained above was repeated for each sample as well]
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
ImageJ: 5 μg/mL sample
ImageJ: 0.5 μg/mL sample
ImageJ: zero DNA
Calibrator Mean Values
Table 1: Calibrator Raw Data
Table 2: Calibrator Means
Calibration curves
Images of Our PCR Negative and Positive Controls
ImageJ: Negative Control PCR sample
ImageJ: Positive Control PCR sample
PCR Results: PCR concentrations solved
PCR Results: Summary
Our positive control PCR result was 24.73 μg/mL.
Our negative control PCR result was -3.55 μg/mL.
Observed results
Patient 87717 : The images for this patient appeared to be very fluorescent green in color. The first product tube (G5 1-1) appeared to be the brightest in color, having the highest initial PCR product concentration of 25.80 micrograms/mL. The second tube had the lowest concentration, 24.21 micrograms/mL, but was the most similar to in color and concentration to the positive control of 24.73 μg/mL.
Patient 56788 : The images for this patient were less fluorescent than the positive control, but were still very similar. The first tube had the lowest concentration of 22.74 μg/mL and appeared to be the least fluorescent image. The last tube had an initial concentration value very similar to the positive control, and the images appeared similar as well.
Conclusions
Patient 87717 : This patient had initial PCR product Concentrations that were very close to that of the positive control, only having a difference of at most 1.08 micrograms/mL from the control value. Therefore it can be concluded that this patient has tested positive for the disease SNP.
Patient 56788 : This patient had initial PCR product Concentrations that were very close to the positive control value, having values that were at most 1.98 micrograms/mL different than the positive control. From this it can be concluded the patient has tested positive for the disease SNP.
BME 100 Fall 2017
