BME100 f2018:Group12 T0800 L5

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Team 12 Name: Karla Sharbuno Role(s) Name: Sean Stanek Role(s) Name: Garrett Moormann Role(s) Name: Bailey Kaufmann Role(s) Name: Alexa Sanchez Role(s) LAB 5 WRITE-UP PCR Reaction Report Before pipetting, all PCR tubes were labeled by Alexa with the pre-assigned labels. Because the labels were difficult to read, our group created an excel map of the tubes as backup. Garrett and Karla added the DNA to the properly labeled tubes, followed by the PCR reaction mix. To set up the positive control, 50 µL of PCR reaction mix and DNA/primer mix were added to an empty tube labeled "positive control". This step was repeated for the negative control and each patient replicate. When pipetting, the pipettor was pushed to the first stop, then released slowly when drawing the solution in, and to the second stop when pushing it out, to ensure that all of the solution was expelled. Using proper pipetting technique is important to ensure no bubbles occur in the solution and that all the solution was drawn up. All tubes resulted in the same amount of liquid (50 µL of DNA and 50 µL of PCR reaction mix), and all liquid was used from the preassigned PCR and DNA tubes. The tips were changed after each transfer to avoid cross-contaminating samples. After the pipetting was completed, the PCR tubes were placed into the OpenPCR machine, which used the given restraints to set up and run the machine. The DNA was initially heated to 95 degrees Celsius for two minutes, almost reaching boiling point. After 30 seconds at 95 degrees, the double bonded DNA helix breaks into two separate single-stranded DNA molecules. The DNA is then annealed at 57 degreees Celsius, at which point the primers attach to the separated strands. Next, the DNA is extended for 30 seconds at 72 degrees Celsius, causing the DNA polymerase to attach to the primers. The final process is holding the DNA at 72 degrees for two minutes and then cooling at 4 degrees C. After this process, the tubes are removed from the OpenPCR machine and stored in the freezer to preserve until Lab D. Fluorimeter Procedure Imaging set-up SmartPhone: Samsung Galaxy S7 We used a pipettor to place a 160 microliter drop of water in the middle of the first two slides, forming a ball shaped drop. The Blue LED switch was used to turn the excitation light on. We adjusted the settings on the smartphone camera to improve the quality of our image. This included turning the flash off, setting white balance to AUTO, increasing exposure and saturation levels, and setting contrast to the lowest setting. The smartphone was positioned at a right angle on the cradle for the best view of the drop. The height of the plastic trays was changed to allow the smartphone to get a side view of the drop. Once the height was set, we moved the smartphone as close as possible to the drop to take pictures, but the distance could not be under 4cm to ensure a clear image. Without moving the smartphone cradle or fluorimeter, our team measured the distance with a ruler between the drop and smartphone. Placing Samples onto the Fluorimeter 1. Using the micropipettor, place an 80 microliter drop of SYBR GREEN 1 in the middle of the first two rows of the slide, creating a ball-shaped drop. Add an 80 microliter sample of a calf thymus solution to the SYBR GREEN 1 drop. 2. Move the slide to align the center of the drop with the blue LED light, from the middle of the black fiber optic fitting on the other side of the drop. 3. Place a desired smartphone by the flourimeter roughly 4 cm away to focus on the drop. 4. Conceal the smartphone and fluorimeter in a lightbox, while keeping one flap of the box open. 5. Set a timer on the smartphone and lower the flap on the lightbox before taking the focused picture of the drop. 6. Remove the 160 µL drop of solution from the slide by siphoning it with the micropipettor and dispose of it into the waste bin within the hazardous waste container. 7. Re-position the slide to the center of the two circles. 8. Repeat the above procedures until all five positions have been used. Data Collection and Analysis Images of High, Low, and Zero Calf Thymus DNA Picture of 5 μg/mL sample. (High) Picture of 0.5 μg/mL sample. (Low) Picture of 0 μg/mL sample. (Zero) 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 MEAN Standard Deviation IMAGE 1 IMAGE 2 IMAGE 3 5 2.5 C-1 21998071 18020610 19386299 19801660 20210000.389 2 1 C-2 17221272 17123065 17693928 17346088 305213.825 1 0.5 C-3 16361710 16692673 16775982 16610122 219126.3664 0.5 0.25 C-4 16228254 17159694 17768047 17051998 775525.2337 0.25 0.125 C-5 14547743 14406787 14509971 14488167 72963.7533 0 0 C-6 3599386 3831514 3798565 3743155 125592.807 Calibration curves Data Plot 1 for Calibration Curve. Data Plot 2 for Calibration Curve. Images of Our PCR Negative and Positive Controls Picture of Negative PCR sample. Picture of Positive PCR sample. 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) G12 P 22898754 2.390698826 12 28.68838592 G12 N 11933718.33 1.103722809 12 13.2446737 G12 1-1 1798457.67 0.9704762523 12 11.64571503 G12 1-2 10241250 0.9050762911 12 10.86091549 G12 1-3 10256129.67 0.9068227312 12 10.88187277 G12 2-1 9779964 0.8509347418 12 10.2112169 G12 2-2 11943561.67 1.10487813 12 13.25853756 G12 2-3 9619301 0.8320775822 12 9.984930986 PCR Results: Summary Our positive control PCR result was 28.7 μg/mL Our negative control PCR result was 13.2 μg/mL Observed results Patient 69455: Observed results of 0.9704762523 μg/mL, 0.9050762911 μg/mL, 0.9068227312 μg/mL showed that there was no green florescence. Patient 1 Sample. Patient 83319: Observed results of 0.8509347418 μg/mL, 1.10487813 μg/mL, 0.8320775822 μg/mL showed evidence of green florescence. Patient 2 Sample. Conclusions Patient 69455: Had no evidence of green florescence and showed inconclusive results. This is because the results were outside the range of 13.2 μg/mL - 28.7 μg/mL. Therefore patient 69455 has no sign of the infection marker that was being observed and tested. Patient 83319: Had evidence of green florescence and showed the closest similarity to the negative control of 13.2 μg/mL. This contradicts our hypothesis in the observed results, stating that the positive control shows evidence of the infection marker. Patient 83319 had no sign of the infection marker, yet displayed the green florescence.