The pre-lab was used to determine how to use the pipettor. This helped make the process easier. We were able to feel the difference between the first stop and the second stop of the pipettor. The final tubes all had the same amount of liquid and there was no liquid left in the original sample tubes. We had two pipettors who were using a micropipettor for the first time and we had an issue with sample 1-3 due to pipetting. The pipettor forgot to fully release the liquid and sucked some of it back into the tip. This could lead to cross contamination as well as error. The same labeling scheme from the previous lab was used. The only difference is that we put the "G11" on the opposite side of tube from the patient ID numbers.
Fluorimeter Procedure
Imaging set-up
We started by opening the box and taking out the unit that holds the slides. We placed a glass slide into the unit with the smooth side of the slide facing down. We then turned on the fluorimeter's light and aligned the circles on the slide with the light beam that the fluorimeter produced. The top of the box that it came in was popped open and the box was flipped over in order to reduce the amount of light hitting the fluorimeter. We then took the piece that held the phone and measured the distance between the phone and the fluorimeter. We set the camera to take 10 pictures. The button for the camera would be pressed, and we would proceed to cover the opening in order to reduce the amount of light inside of the container.
Placing Samples onto the Fluorimeter
Step One: Lift the box so that the fluorimeter is out in the open
Step Two: Take the glass slide and slide it into the groove of the fluorimeter with the smooth side of the slide down.
Step Three: Take the SYBR Green in the pipette and center the tip to the center of the circle on the glass slide. Release the liquid.
Step Four: Take the DNA sample in the pipette and proceed to put the liquid into the ball of SYBR Green already on the slide.
Step Five: Realign the slide so that the ball of liquid is perfectly centered with the fluorimeter's beam of light.
Step Six: Put the box back over the fluorimeter and proceed to take the pictures needed.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
Calibrator Mean Values
Initial Concentration of 2X Calf Thymus DNA Solution (μg/mL)
Final DNA Concentration in SYBR Green I Solution (μg/mL)
Our positive control PCR result was 55.92887648 μg/mL
Our negative control PCR result was -3.426557992 μg/mL
Observed results
Patient 75247 (1) : The initial PCR product concentrations found were -7.451277912 μg/mL, 1.721952674 μg/mL, and 6.862548343 μg/mL for the three replicates. Using this data, the mean concentration is .377741 μg/mL. The images taken of the drop in the fluorimeter show drops that are, for the most part, half light and half dark. Though some of the images are not half and half, all images taken of the drops for this patient have a completely dark area in the drop along with a lighter area.
Patient 55425 (2): The initial PCR product Concentrations found were 23.31412355 μg/mL, 20.64586778 μg/mL, and 12.70040376 μg/mL for the three replicates. Using this data, the mean concentration is 18.88679836 μg/mL. In the fluorimeter images, the entire drop is illuminated to some degree. There are also areas that are completely white in the drop. There is no area of complete black in the drops.
Conclusions
Patient 75247 : The diseased DNA concentration in the positive control is 55.92887648 μg/mL and the negative control concentration is -3.426557992 μg/mL. The 1-1 replicate, like the negative control, contains a negative value for the concentration. The other replicates for this patient are both below 10 μg/mL. Two of the three replicates have concentrations that are below 2 μg/mL. Because the individual concentrations and the mean are so low, it appears that Patient 75247 is negative for the disease. Due to the fact that this patient has the only negative concentration outside of the negative control, it is clear that the results are negative.
Patient 55425: The diseased DNA concentration in the positive control is 55.92887648 μg/mL and the negative control concentration is -3.426557992 μg/mL. The results for this patient are not numerically close to either the positive or negative controls. There was not a single replicate with a negative concentration like that of the negative control. The mean for the concentrations is almost 20, which is still far from the positive control value. Based on the fact that the concentration is clearly positive, the negative result can be eliminated. It is difficult to verify that the result is positive because of the numerical difference. It is clear that the result is positive when comparing this patient's images to the images for the positive control. The drops for the patient and the positive control lit up with no fully black areas. Based on both numerical and photographic results, Patient 55425 is positive for the disease.
BME 100 Fall 2018
