Name: Michael Finocchiaro
LAB 5 WRITE-UP
PCR Reaction Report
Before the experiment the group went over the pre lab reading and the video on the functions of the micropipettor. The video was especially helpful when it came to determining where the first and second stop on the micropipette were, knowing how to prevent bubbles and setting the correct amount of microliters. One member of the group also had previous micropipetting experience which helped immensely when it came down to using the micropipette. The proper materials were gathered before the experiment which included: the patient DNA samples, the micropipette, the PCR reaction mix, and the empty PCR tubes. The empty tubes were labeled positive control, negative control, 1-1, 1-2, 1-3, 2-1, 2-2, and 2-3. The experiment started with putting 50 microliters of PCR reaction mix into the empty positive control tube. Then 50 microliters of the positive control were added to the same tube. This lead to 100 microliters of total solution. These steps were repeated with the negative control, with patient 1's DNA and patient 2's DNA. Once all the solutions were put together correctly, the 8 solutions were placed into the PCR machine. The final reactions ended up all containing 100 microliters of each solution.
To properly calibrate the iPhone that was used to capture the images of the solutions, a 160 microliter drop of water was used as a point of reference. This drop was placed on the rough side of the slide which was then inserted onto the fluorimeter and the iPhone was placed on a stand. The fluorimeter was elevated by three plastic trays so that it was held level with the camera. Once this was all set up, it was determined that the correct distance between the iPhone and the fluorimeter for a focused photo was 8cm.
Placing Samples onto the Fluorimeter
- Place a slide with the rough side facing up onto the fluorimeter
- Set the micropipettor to 80 microliters
- Place 80 microliters of SYBR Green I on the rough side of the slide between the first two dots
- Place 80 microliters of the solution being tested onto the drop of SYBR Green I
- Align the drop with the light on the fluorimeter
- Set the iPhone timer to 3 seconds and ensure the iPhone is 8cm from the fluorimeter
- Place the black box over both the iPhone and fluorimeter
- Activate the camera and lower the flap of the box
- Remove the black box and repeat these steps for all trials
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 soulution (micrograms/mL)
(The Calibration Curves do have the error bars placed on the data points, but most of them are too small to be visible on the scale of this graph.)
Images of Our PCR Negative and Positive Controls
PCR Results: PCR concentrations solved
|PCR Product TUBE LABEL
||MEAN (of RAWINTDEN DROP - BACKGROUND)
||PCR Produce Concentration (micrograms/mL) (Step 5 calculation)
||Initial PCR Product Concentration (micrograms/mL) (Step 6 calculation)
PCR Results: Summary
- Our positive control PCR result was 27.564 μg/mL
- Our negative control PCR result was 4.44 μg/mL
- Patient 83982 : Patient 83982's drops did not visibly glow green when placed on the fluorimeter. The samples looked similar to the negative control in the images. The initial concentrations for the three samples were found to be 1.464 μg/mL, 1.2 μg/mL, and 3.12 μg/mL, respectively.
- Patient 29244 : Patient 29244's drops glowed a vibrant green color when placed on the fluorimeter. These samples looked extremely similar to the positive control in the images. The initial concentrations for the three samples were found to be 38.508 μg/mL, 37.332 μg/mL, and 38.1 μg/mL, respectively.
- Patient 83982 : Since all three initial concentrations of the samples for Patient 83982 (1.464 μg/mL, 1.2 μg/mL, and 3.12 μg/mL) were found to be below the threshold of the negative control's initial concentration of 4.44 μg/mL, it can be concluded that Patient 83982 is negative for the disease.
- Patient 29244 : Since all three initial concentrations of the samples for Patient 29244 (38.508 μg/mL, 37.332 μg/mL, and 38.1 μg/mL) were found to be above the threshold of the positive control's initial concentration of 27.564 μg/mL, it can be concluded that Patient 29244 is positive for the disease.