LAB 5 WRITE-UP
PCR Reaction Report
The pre-lab reading was helpful to understand just how the lab needed to be setup. A few of us have experience with using the micropipettors, so we knew what the first and second stops meant already. There was some liquid left in the tubes of the DNA samples and the PCR reaction mix, and one of the reactions had slightly less liquid in it than the rest. The labeling scheme worked fine and didn't need to be changed.
Web camera set-up
For this lab a phone camera was used instead of a webcam. The phone was placed in the cradle standing nearly vertically. The flourimeter was then placed on some small plates to lift it up to the camera. The height of the flourimeter was adjusted using more or less plates so that the camera could take a picture directly on the side of the drop. The camera was then set to use a timer.
Placing Samples onto the Fluorimeter
- With gloves, find the smooth side of the slide and place it on the flourimeter smooth side down.
- Set the camera in the cradle and set the timer on it.
- Change the height of the flourimeter so the camera can take a picture of the side of the drop of the solution.
- Use 80 microliters of SYBR Green I solution and place a drop on the first 2 clear circles in the middle of the slide.
- Now place 80 microliters of the sample/calibration solution on top of the SYBR Green I drop.
- Move the slide so that the light from the flourimeter illuminates the middle of the drop and focuses on the other side.
- Change the distance of the camera so that it takes a close picture of the drop and remains in focus, then record that distance.
- Cover the setup with the lightbox and make sure the drop is in focus.
- Now press the timer on the camera and close the box with the other flap.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
Calibrator Mean Values
|Initial Concentration of 2X Calf Thymus DNA solution (micrograms/mL)
||Final DNA concentration in SYBR Green I solution (µg/mL)
||RAWINTDEN DROP - BACKGROUND
Images of Our PCR Negative and Positive Controls
PCR Results: PCR concentrations solved
|PCR Product TUBE LABEL
||MEAN (of RAWINTDEN DROP - BACKGROUND)
||PCR Product Concentration (µg /mL)
||Initial PCR Product Concentration (µg /mL)
| G11 +
| G11 -
| G11 1-1
| G11 1-2
| G11 1-3
| G11 2-1
| G11 2-2
| G11 2-3
PCR Results: Summary
- Our positive control PCR result was -3.866892571 μg/mL
- Our negative control PCR result was -8.648745143 μg/mL
- Patient 87806 : The image showed a drops that were faintly illuminated. It also had an average of -7.013 μg/mL.
- Patient 99993 : The images for his patient were not illuminated and had an average of -7.679 μg/mL.
- Patient 87806 : This patient returned a negative results because its PCR results most resembled the negative control.
- Patient 99993 : This patient also returned a negative result for the same reason, its PCR results also resembled the negative control.