LAB 5 WRITE-UP
PCR Reaction Report
The pre-lab materials provided were helpful in terms of being able to complete the lab. The micropipetting video tutorial covered everything one needed to know about pipetting, and the examples of what not to do helped to understand how one mistake could lead to an error in our results. We learned that the first stop on the pipette is used to pick up the desired liquid and the the second stop is used to deposit the liquid into the desired location. At times, there was liquid left in the tubes. What we could have done to prevent this was set the pipette to a higher volume in order to ensure that all of the liquid was picked up. The labeling scheme we used was effective because it was simple, yet it was easy to detect which tube contained which solutions and when placed into the OpenPCR machine, it was easy to detect which tubes were ours.
Smart Phone Camera Settings
- Type of Smartphone: IPhone 6
- Flash: Off
- ISO setting: Automatic
- White Balance: Automatic
- Exposure: Automatic
- Saturation: Automatic
- Contrast: Automatic
Camera Set Up
- In order to set up the smart phone camera to accurately take a picture of the drop sideways, we vertically inserted the smart phone in a cradle. To stabilize the phone, we placed a plastic box underneath the cradle. To adjust the height of the fluorimeter, we also placed a plastic box under it. Then, we measured the distance between the smart phone cradle and the drop.
- Distance between the smart phone cradle and drop: 8 centimeters
Placing Samples onto the Fluorimeter
- To calibrate the fluorimeter, use a micropipette to place 160 μL of water in the middle of the first two rows of the slide.
- Turn on the LED light on the fluorimeter. If needed, adjust the slide so that the light passes through the drop of water.
- Place the camera the distance that was measured above in order to focus the camera. In this case, 8 centimeters.
- Pick up the 160 μL of water with a micropipette and discard of it in the liquid waste cup.
- Place 80 μL of SYBR Green I in the middle of the second and third rows of the slide.
- On the same drop of SYBR Green I, place 80 μL of the first concentration of the calf thymus solution.
- Adjust the slide so that the light passes through the drop of water.
- Focus the smart phone camera. Make sure the timer is on for 3 seconds. Capture a picture of the drop. When pressing the capture button, quickly close the lid of the light box in order to remove as much stray light as possible.
- Repeat the previous step two more times in order to have a total of three pictures of the drop.
- Remove the light box carefully as to not move the phone cradle.
- Use the micropipette to remove the 160 μL of the solution. Dispose of it in the liquid waste cup.
- Repeat steps 5-11 with the remaining concentrations of the calf thymus DNA solution. Make sure to capture three pictures of each.
- To create the solutions that will be tested using the PCR reaction samples, transfer 100 μL from the PCR tubes into a 500 μL tube of buffer and invert the tube to mix it. Make sure to label the buffer tube with what PCR solution is being transferred into it.
- Repeat steps 5-11 to test the 8 tubes containing the buffer + PCR solution on the fluorimeter.
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 solution (µg/mL) || Sample Number || RAWINTDEN DROP - BACKGROUND (Image 1) || RAWINTDEN DROP - BACKGROUND (Image 2) || RAWINTDEN DROP - BACKGROUND (Image 3) || Mean || Standard Deviation
| 5 || 2.5 || C-1 || 5322561 || 5603115 || 5284249 || 5403308.333 || 174094.7454
| 2 || 1 || C-2 || 3336421 || 3296463 || 2929210 || 3187364.667 || 224459.427
| 1 || 0.5 || C-3 || 2909402 || 2510609 || 2819311 || 2746440.667 || 209144.7545
| 0.5 || 0.25 || C-4 || 2361630 || 2034078 || 2466779 || 2287495.667 || 225675.5711
| 0.25 || 0.125 || C-5 || 2358973 || 2158973 || 2180603 || 2232849.667 || 109760.1277
| 0 || 0 || C-6 || 2017278 || 1958184 || 2053455 || 2009639 || 48092.6876
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)(Step 5 calculation) || Total Dilution || Initial PCR Product Concentration (µg /mL)(Step 6 calculation)
| G2 P || 8048518.333 || 8.89 || 12 || 106.68
| G2 N || 940644 || -1.56 || 12 || -18.72
| G2 1-1 || 2168998 || 0.248 || 12 || 2.976
|G2 1-2 || 2429379 || 0.631 || 12 || 7.572
| G2 1-3 || 3535750 || 2.26 || 12 || 27.12
| G2 2-1 || 5043321 || 4.47 || 12 || 53.64
|G2 2-2 || 6556006 || 6.64 || 12 || 79.68
| G2 2-3 || 6488055 || 6.59 || 12 || 79.08
PCR Results: Summary
- Our positive control PCR result was 8.89 μg/mL
- Our negative control PCR result was -1.56 μg/mL
- Patient 63690 : The DNA was green and circular. The Initial PCR concentrations were closer to the positive control
- Patient 22568 : The DNA was green and circular. The Initial PCR concentrations were closer to the negative control.
- Patient 63690 : Positive because the majority of the initial PCR concentrations were closer to that of the positive control.
- Patient 22568 : Negative because the majority of the initial PCR concentrations were closer to that of the negative control.