LAB 5 WRITE-UP
PCR Reaction Report
In our group, Midori has previously used a micropipette in an internship she had recently so she throughly understood the procedure involved with using a micropipette. For the rest of the lab group, the pre-lab video and interaction really helped us understand the process of micro pipetting. Before using the micropipette the first portion of the lab, we practiced using the micropipette to get a feel of the first and second stops. There were a few errors within the micro pipetting process in our lab because not all of our final reactions contained the exact same amount of liquid. When mixing the DNA samples and the PCR reaction mix, there was not any liquid left over after in the tubes. To label the tubes, we followed the general format as the table given by labeling our group number (G3) along with the patient ID (1-1, 1-2, 1-3, 2-1, etc.).
To set up our device to capture images from the fluorimeter, we stacked a couple of empty plastic trays to lift the fluorimeter to an appropriate level for the camera we were using. Then, we roughly measured the distance of the camera lens to the location of where the droplets would go on the slide to about four centimeters away. We placed an iPhone into the tray so that it could be stabilized and could take clear pictures of the droplets. We placed a slide into the fluorimeter and after placing each droplets would place the black box over the top of the device to take the picture.
Placing Samples onto the Fluorimeter
- While wearing gloves, identify which side of the slide is the smooth glass back.
- Place the fluorimeter on the table and turn it on.
- Once the fluorimeter is on, place the smooth side of the slide downwards and slide it into the fluorimeter.
- Open the camera app on the smartphone, set the camera timer for three seconds, and place the phone on the cradle.
- If needed, adjust the height of the fluorimeter so that the camera view of the slide is nearly edge on.
- Using the micropipette, we placed 80 microliters of the SYBER green solution on the slide.
- Using the pipette with a new tip, measure 80 microliters of the sample/calibration solution and inject it to the top of the Cyber Green I drop.
- Next, the slide must be adjusted so that the light is directly pointed in the center of the drop and the drop focuses the light on the other side.
- Then, place the smartphone four centimeters away from the fluorimeter, making sure that view of the drop is close and focused.
- Place the lightbox over the fluorimeter and the smartphone and keeping one flap up.
- Double check that the drop is focused on the camera.
- Set the smartphone to take a picture in three seconds, and close the flap before the picture is taken.
- Once the picture is taken, remove the 160 microliters from the slide and dispose the liquid in a liquid waste container.
- Move the slide so the light is pointing to the next two circles on the slide.
- Repeat steps 1-14 until all five possible measurement positions of the slide is used.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
5 μg/mL Sample
0.25 μg/mL Sample
0 μg/mL Sample
Calibrator Mean Values
Images of Our PCR Negative and Positive Controls
PCR Results: PCR concentrations solved
PCR Results: Summary
- Our positive control PCR result was 0.1328μg/mL
- Our negative control PCR result was 0.2991μg/mL
- Patient _(1)_73066___ : The drop in the image was clear. This meant that the patient's results had been negative.
- Patient _(2)_57254___ : The drop in the image was a glowing greenish color. This meant that the patient's results had been positive.
- Patient _(1)_73066___ :Negative. Our group concluded that patient one was negative because the values matched our negative control.
- Patient _(2)_57254___:Positive. Our group concluded that patient two was positive because the values matched our positive control.