LAB 5 WRITE-UP
PCR Reaction Report
Our pipetting of the samples was successful during the setup of this laboratory procedure. The appropriate amounts of DNA sample liquids and PCR reaction mix were added to the tubes with no observable error. The tubes were labeled as we had previously planned in the earlier portion of this lab, and we successfully transferred the liquids between the tubes without contamination.The pre-lab provided appropriate information about how to complete a PCR lab. Through the interactive activities, we were able to familiarize ourselves with the important and significant procedure. In addition, the reading provided informative background descriptions and definitions that helped in the progression and completion of the laboratory procedure. The first and second stops on the pipettor were easy to distinguish after the first few pipetting steps. When you hit the first stop on the pipettor, it is quite difficult to go past it accidentally. Therefore, when you purposefully expel the sample into the PCR tube, you can perceive when it is fully expelled at the second stop. The final reactions held the same amount of liquid as the original components combined. Precision was used in transferring the components into the reaction, and therefore, there was the exact same amount of liquid. There were no component liquids left in the tubes of DNA and the PCR reaction mix. Since precision was used in removing these liquids, there was no liquid left in these tubes and all was added into the PCR tubes. We used our original labeling scheme from Part A for the procedure in Part C. We saw no need to change it, given that it was unique to our group seeing as we labeled our PCR tubes with “G4.”
Smart Phone Camera Settings
- Type of Smartphone: HTC 1
- Flash: off
- ISO setting: 1600
- White Balance: auto
- Exposure: 2x
- Saturation: 2x
- Contrast: -2x
The phone, with camera included, was placed in the phone cradle in front of the fluorimeter. Once the drop was set on the slide, the camera was shifted to a closer distance while retaining the focus on the drop.
- Distance between the smart phone cradle and drop (beam on fluorimeter) = 7 centimeters
Placing Samples onto the Fluorimeter
- Place the smooth side of the slide downward
- After setting the camera timer to three seconds, place it in the provided cradle
- Change the height of the fluorimeter to get a side view of the drop on the camera
- In the middle of the slide, put an eighty microliter drop of SYBR Green 1 upon the first two clear circles.
- Then, put an eighty microliter drop of the sample on this drop.
- Shift the slide so that the light shines through the drop, focusing the light
- After adjusting the distance between the camera cradle and fluorimeter so that the images are close, yet focused, record this distance
- Place the lightbox over the set up with one flap open
- Set the timer and close the flap before it captures the image
- Place the drop in the waster container after recording which sample was just pictured
- Next, slide the slide into the next position and repeat the experiment for all the possible slide posititons
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 || 1236527 || 1188644 || 1275005 || 1233392 || 43265.76879
| 2 || 1 || C-2 || 1094773 || 1284966 || 1424882 || 1268207 || 165691.3879
| 1 || 0.5 || C-3 || 1413420 || 920830 || 900279 || 1078176 || 290511.3129
| 0.5 || 0.25 || C-4 || 986848 || 1150559 || 1056754 || 1064720 || 82145.72253
| 0.25 || 0.125 || C-5 || 887658 || 899227 || 936575 || 907820 || 25565.56197
| 0 || 0 || C-6 || 872434 || 1119336 || 93454 || 695074.7 || 535444.4129
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 (μL/mL) (Step 5 calculation) || Total Dilution || Initial PCR Product Concentration (μL/mL) (Step 6 Calculations)
| G4 + || 15179.6667 || -3.27514 || 12 || -39.30169
| G4 - || 18965 || -3.25971 || 12 || -39.11656
| G4 1-1 || 3783.33333 || -3.32159 || 12 || -39.85905
| G4 1-2 || -18287 || -3.41154 || 12 || -40.93845
| G4 1-3 || 44859.3333 || -3.15417 || 12 || -37.85014
| G4 2-1 || 48412 || -3.13970 || 12 || -37.67639
| G4 2-2 || 65845 || -3.06865 || 12 || -36.82379
| G4 2-3 || 67970.3333 || -3.05999 || 12 || -36.71985
PCR Results: Summary
- Our positive control PCR result was -39.85904965, -40.9384463, and -37.8501404 μg/mL.
- Our negative control PCR result was -37.67638968, -36.82379169, and -36.71984774 μg/mL.
- Patient 12188 : Quantitatively, the values for the initial PCR reaction concentration were very close to both the negative and positive control values at at approximately -39 micrograms per milliliter. Qualitatively, the patient samples looked identical to the negative control; the pictures were very dark except for the poles of the drops and the beam of light.
- Patient 54737 : Quantitatively, the initial PCR reaction concentration values were close to the negative and positive control amounts at about -36 micrograms per milliliter. Qualitatively, the patient samples looked exactly like the negative control; additionally, the photos were very dark except at the poles of the drops and the beams of light.
- Patient 12188 : For samples one and two, the resulting values of the initial PCR product concentration were closer to the value of the positive control initial PCR product concentration. The third sample was closer to the negative control initial PCR concentration than to the positive control value. Therefore, since two out of the three samples were closer to the positive control, patient 12188 was positive.
- Patient 54737 : For all three samples, the resulting values of the initial PCR product concentration were closer to the value of the negative control than to the positive control of the initial PCR product concentration. Therefore, since all three samples were closer to the negative control, patient 54737 was negative.