LAB 5 WRITE-UP
PCR Reaction Report
Our experience with micro pipetting was rewarding. The tutorials and pre lab assignments allowed us to prepare for this lab and we were able to comply to each of the procedural steps associated with micro pipetting. We were able to understand the difference between the first and second stop on the pipette allowing us to avoid problems or inconsistencies in our mixtures. Each of the final reactions have the same amount of liquid and there was no remaining liquid in the tubs where we acquired the DNA and PCR mixture. We did not have to change the labeling of our tubes. We labeled our tubes with a black marker on the side of the tub. In total we were able to enjoy ourselves during the lab and we were able to utilize what we had learned to properly us a micro pipette.
Smart Phone Camera Settings
- Type of Smartphone: LG G4
- Flash: off
- ISO setting: 2700
- White Balance: Automatic
- Exposure: High
- Saturation: High
- Contrast: Low
- Distance between the smart phone cradle and drop = 5 cm
Placing Samples onto the Fluorimeter
- Wear gloves and a lab coat.
- Place camera with pre set settings on cradle and set the timer to 3 seconds.
- Use a plastic tray to adjust the fluorimeter so the camera lens faces the drop face on.
- Place a slide inside the fluorimeter with the rough part of the glass pointing up.
- Using a ruler to record the distance between the camera and the first 2 rows of the slide inside the fluorimeter to a close distance of 4 cm or greater.
- Turn on the switch for the Blue LED light.
- Using a mircopipetter, place 80μL of the SYBR GREEN I solution in the first 2 rows of the slide.
- Place 80 μL on one of the calf thymus solutions provided on top of the SYBR GREEN drop to create 1 sample drop.
- Make sure that the slide is adjusted, so the Blue LED light goes through the center of the sample drop on to the other side.
- Take three images of the sample drop making sure it is focused and that the light box covers as much light as possible on each take.
- Carefully remove the box without moving the smartphone of set up. If any movement occurs, make sure to set it up to the original distance.
- Remove the sample drop using the micropipetter and discard of the sample in the appropriate container.
- Repeat steps 5- 10 for the remaining calf thymus solutions provided, making sure to either move the slide to a new set of rows or use a brand new slide.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
Image of High Calf Thymus DNA
Image of Low Calf Thymus DNA
Image of 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
| ||Image 1||Image 2||Image 3||
Images of Our PCR Negative and Positive Controls
PCR Positive Control
PCR Negative Control
PCR Results: PCR concentrations solved
|PCR Product TUBE LABEL
||MEAN (of RAWINTDEN DROP - BACKGROUND)
||"PCR Product Concentration (µg /mL)|
(Step 5 calculation)
||"Initial PCR Product Concentration |
(Step 6 calculation)"
| G8 +||53719487||76199145||12||914389740
| G8 -||22094584||23490973||12||281891680
| G8 1-1||19099824||18499707||12||221996480
| G8 1-2||40015513||53359188||12||640310260
| G8 1-3||31971056||39951760||12||479421120
| G8 2-1||37814774||49691290||12||596295480
| G8 2-2||41920503||56534172||12||678410060
| G8 2-3||38059206||50098677||12||601184120
PCR Results: Summary
- Our positive control PCR result was 914389740 μg/mL
- Our negative control PCR result was 281891680 μg/mL
- Patient 27435 : The droplet did not have any visible green. ImeageJ was not able to see as much SYBR green when analyzing the pictures of the droplets. The average SYBR green that was exhibited in the samples was 447242620 μg/mL
- Patient 16764 : The droplet did not have any visible green. ImageJ was able to analyze the droplet and showed that there was a minute amount of green inside the droplet that we did not initially see. The average of SYBR green was 625296553.3 μg/mL
- Patient 27435 : Negative
- Patient 16764 : Positive
We found that based on the results of the PCR product concentrations that patient 16764 was Positive and patient 27435 was Negative. We came to this conclusion by comparing the PCR product concentrations with that of the Positive and Negative controls. Two out of the three samples were negative and therefore we concluded that the patient was negative. Patient 27435 was three out of three for the Positive control and came to the conclusion that the patient was indeed positive.