LAB 5 WRITE-UP
PCR Reaction Report
Since, the whole team had a really good understanding of how the micropipette works, the team had a really good experience manipulating this lab equipment.The pre- labs and the readings were helpful because they briefly described and introduced the team to the material that was about to be covered by the instructor.The mechanism of the pipettor was completely understood by the whole team, so it was easy to know the difference of the first and the second stop.Our final reaction did had the same amount of liquid in the tubes, and no liquid left in the test tubes that contain the DNA samples and the PCR reaction mix. We did not have to change the labeling scheme of our test tubes.
When setting up the fluorimeter and phone to get adequate photos, first the phone being used to take photos needed to be raised. We stacked 2 pipette tip containers and rested the phone on top. We then set up the fluorimeter about 4-6 centimeters from the point of focus. Next, we made sure the whole set up would fit underneath the black box and tested to make sure the flap would open and close effectively. After tweaking the design a few times to ensure good photos we conducted the trials.
Placing Samples onto the Fluorimeter
- Step one: Using gloves, identify which side was smooth and which was rough
- Step two: Turn on the Fluorimeter and line up the first two divots with the light
- Step three: Using a micropipette transfer 160 microliters of water to the first two divots, this is to calibrate the Fluorimeter
- Step four: Put the iPhone camera on a timer, start it, and close the box and wait for phone to take a photo
- Step five: Using a new tip remove the 160 microliters of water from the first two divots
- Step six: Move the slide so the next two divots line up with the Fluorimeter and place 80 microliters of SYBR Green using a fresh micropipette tip to the slide.
- Step 7: Put the iPhone camera on a timer, start it, and close the box and wait for phone to take a photo (REPEAT 3 TIMES FOR 3 SEPARATE PHOTOS)
- Step 8: REPEAT STEPS 5-7 WITH ALL NECESSARY CONCENTRATIONS
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
5 μg/mL DNA Sample
0.5 μg/mL DNA Sample
0 μg/mL DNA Sample
Calibrator Mean Values
|Initial Concentration of 2X Calf Thymus DNA Solution (μg/mL)||Final DNA Concentration in SYBR Green I Solution (μg/mL)||Sample Number||RAWINTENDEN DROP- BACKGROUND |
(IMAGE 1, IMAGE 2, IMAGE 3)
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) || Total Dilution || Initial PCR Product Concentration (µg /mL) ||
|G14 + ||23589122.33 ||-798.813005 ||12 || -9585.75606 ||
|G14 - ||11858546.33 ||-392.5733597 ||12 || -4710.880316 ||
|G14 1-1 ||9302393 ||-304.0516346 ||12 || -3648.619615 ||
|G14 1-2 ||12414422.67 ||-411.8238214 ||12|| -4941.885857 ||
|G14 1-3 ||8998444.667 ||-293.5256499 ||12|| -3522.307799 ||
|G14 2-1 ||39829061.67 ||-1361.215669 ||12|| -16334.58803 ||
|G14 2-2 ||20563469 || -694.0321028 || 12|| -8328.385233 ||
|G14 2-3 ||29014846.33 || -986.7103592 || 12 || -11840.52431 ||
PCR Results: Summary
- Our positive control PCR result was -9585.75606 μg/mL
- Our negative control PCR result was -4710.880316 μg/mL
- Patient 15727 : The images of this patient had a colorless look and an average initial PCR product concentration of -4037.60 μg/mL.
- Patient 52298 : The images of this patient had a bright green neon glow and an average initial PCR product concentration of -12167.83 μg/mL.
- Patient 15727 : This patient was concluded to be negative due to resembling the negative control value. The negative control value had a value of -4710.88 μg/mL, and the three tests that were administered had an average of -4037.60. This value is much closer to the negative control value than the positive control value (-9585.76 μg/mL). Due to these results, the patient was determined to be negative for the presence of the DNA.
- Patient 52298 : This patient was concluded to be positive due to resembling the positive control value. The positive control value had a value of -9585.76 μg/mL, and the three tests that were taken had an average of -12167.83 μg/mL. This value is closer to the positive control value than the negative control value (-4710.88 μg/mL). Due to these results, the patient was determined to be positive for the presence of the DNA.