LAB 5 WRITE-UP
PCR Reaction Report
The lab group first micropipetted 50 microliters (µL) of each individual DNA/primer mix into properly labeled tubes. Taking care not to cross contaminate each sample mix, another 50 µL of PCR reaction mix was added and mixed with the DNA/primer solutions. Pipette tips were carefully disposed before each individual solution. During the first three mixes, the micropipetter was new to the technique and could have been a source of error for measurement.
These tubes were then placed in the PCR machine and run through 25 cycles. The lid was set to 100°C. The initial step was set to 95°C for two minutes. The cycles were set for 30 seconds each to denature at 95°C, anneal at 57°C and extend at 72°C. The computer connected to the PCR machine logged out the user after 10 minutes, and although this did not appear to affect the machine it could have caused error or a glitch in the cycles.
A few members in our group have had experience with pipetting so they were able to help everyone during the lab. The pre-lab helped because the simulation for the pre-lab was very clear and detailed enough to help understand what we would be doing in the lab. We understood the difference between the first and second stop on the pipettor very easily because of the pre-lab simulation. We used it as guidance during the lab. The final reaction did not have the same amount of liquid due to air bubbles in the solution and transfer process, most likely caused by human error. Additionally, there was not any excess liquid left in the tubes that the DNA samples and PCR reaction were contained in. We did not have to adjust or change the labeling system that we established in the previous lab.
To properly capture images from the fluorimeter, a smartphone camera needs to be set up. To make sure the Camera is at the same level of the beam, try adding support underneath the device to raise it. Click on the timer on the camera app and set the timer to at least 3 seconds. Capture each image after lowering down the end flap to eliminate any exterior light coming through.
Placing Samples onto the Fluorimeter
- [Step one: Find the smooth side of the slide. ]
- [Step two:Turn on the Fluorimeter and place the slide in the device smooth side down. ]
- [Step three: Set up the camera with a 3-second timer on the cradle and adjust the height of the device to make sure the droplet is in perfect view.]
- [Step Four: Place 80 uL drop of SYBR green solution in the middle of the slide towards the top edge (first two bubbles). ]
- [Step Five: Place 80 uL drop of Calibration sample on top of the SYBR green solution. Make sure the slide is adjusted so the droplet is in the center of the beam. ]
- [Step Six: Record the distance of the Camera from the Device. Cover the Device with the black box, with one flap open for easy access. ]
- [Step Seven: Take a picture and close the end flap before the shutter takes the snapshots. ]
- [Step Eight: After taking 3 different pictures, remove the 160 uL solution and dispose in the liquid disposal. Move the slide up to the next row of circles. ]
- [Step Nine: Repeat the above steps until you have used all 5 measurement positions on the slide. ]
Data Collection and Analysis
Image of High Calf Thymus DNA
Image of Low Calf Thymus DNA
Image of Zero Calf Thymus
Calibrator Mean Values
||Final DNA concentration in SYBR Green I solution (µg/mL)
||Mean Pixel Value
||RAWINTDEN OF THE DROP
||RAW INTDEN OF THE BACKGROUND
Images of Our PCR Negative and Positive Controls
Image of Positive Control
Image of Negative Control
PCR Results: PCR concentrations solved
|PCR Product Concentration (µg /mL)
||"Initial PCR Product Concentration
| (µg /mL)
PCR Results: Summary
- Our positive control PCR result was 53.120 μg/mL
- Our negative control PCR result was 8.010 μg/mL
- Patient 27468: After observing the droplet under the ImageJ software, we noticed a strong appearance of green towards the center of the drop. 4.17 μg/mL was observed for Patient 27468.
- Patient 96944: While observing the droplet during the experiment, The drop for Patient 96944 showed no difference in appearance (naked eye observation) as compared to the Patient 27468 droplet. 25.16 μg/mL was observed for Patient 96944.
- Patient 27468: Negative___: We concluded the patient was negative based on the values that matched our negative control.
- Patient 96944:Negative___: We concluded the patient was negative based on the values that matched our negative control.