BME100 f2018:Group10 T1030 L5

BME 100 Fall 2018	Home People Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3 Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6 Course Logistics For Instructors Photos Wiki Editing Help
OUR TEAM Error creating thumbnail: File missing Name:Talia Hertzberg Error creating thumbnail: File missing Name:Samuel Whitworth Error creating thumbnail: File missing Name:Lauren Everett Error creating thumbnail: File missing Name:Maritza Trejo Error creating thumbnail: File missing Name:Angel Cardenas LAB 5 WRITE-UP PCR Reaction Report Out of the entire group Lauren and Talia were the only ones to have used micropipettors before. To help everyone feel more comfortable, Lauren and Talia worked with pipetting the solutions into the proper test tubes. The pre-lab reading definitely helped refresh the steps and made it easier to carry out the proper procedure. We did know the difference between the first and second stops after testing out the micropipettor a couple times. From observations, each test tube seemed to have as close of a reading as possible to the 100 μl. In the DNA sample test tubes and PCR reaction mix test tubes, there was a little bit of solution left in them. Our labeling scheme was our group number, then the labels of; positive, negative, patient 1-1, patient 1-2, patient 1-3, patient 2-1, patient 2-2, patient 2-3. While pipetting, we did not need to change our scheme because it worked out perfectly. Fluorimeter Procedure Imaging set-up The materials that we gathered were a fluorimeter, about 10 glass slides, a phone stand for the smartphone, and a light box.We used our teammate's iPhone, of which required us to raise the light box up with about 4 carton boxes so that it was properly aligned with the camera on the phone. We also made sure that the phone was close enough to the droplet to get a clear image, so we remained about 4 cm away from the drop.This allowed us to see the appropriate solution of our PCR reaction mixed with the SYBR Green 1 solution on the glass slide. Then, the phone was set to have a 3 second timer for taking the pictures and the flash was turned off. So when the pictures needed to be taken, we just pressed the capture button on the phone and quickly lowered the lid of the light box. Then, our pictures were labeled with the right trial and saved to the phone to be later uploaded on ImageJ. Placing Samples onto the Fluorimeter 1. Place a 80 microliter drop of SYBR Green 1 on top of the 80 microliter drop of one of the calf thymus solutions (these drops will be placed in the middle of the first 2 rows of the slide) 2. Align the drop so that the blue LED light is focused from one side of the drop to the other 3. Place phone on stand and press capture button while immediately closing lid of light box 4. Take 3 pictures of the droplet and make sure the flash is off and the image is focused 5. Remove the box. Be careful to not adjust the position of the phone too much 6. Take micropipettor and remove the 160 microliter drop from the slide and prepare for new sample (you can always take a new slide if needed) 7. Repeat steps 1-6 for all the other concentrations of calf thymus solution Data Collection and Analysis Images of High, Low, and Zero Calf Thymus DNA Error creating thumbnail: File missing 5 μg/mL sample Error creating thumbnail: File missing 0.5 μg/mL sample Error creating thumbnail: File missing 0 μg/mL sample 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 RAWINTDEN DROP - BACKGROUND RAWINTDEN DROP - BACKGROUND MEAN Standard Deviation Image 1 Image 2 Image 3 5 2.5 C-1 19607443 27267726 26689551 24521573.33 4265569.046 2 1 C-2 15769374 15861018 165006363 65545585 86135572.62 1 0.5 C-3 16840698 14789405 17830034 16486712.33 1550914.43 0.5 0.25 C-4 16626467 16080101 15705150 16137239.33 463308.5856 0.25 0.125 C-5 12399828 12182004 11214249 11932027 631083.0109 0 0 C-6 5559981 6217025 6217025 5998010.333 379344.5303 Calibration curves Error creating thumbnail: File missing Error creating thumbnail: File missing Images of Our PCR Negative and Positive Controls Error creating thumbnail: File missing Positive PCR Mix Negative PCR Mix 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) G10:Positive 16662422 0.4887474 12 5.8649688 G10:Negative 4070948.333 0.0690316111 12 0.8283793332 G10:1-1 26116407.33 0.8038802443 12 9.646562932 G10:1-2 19016194.67 0.567206489 12 6.806477868 G10:1-3 15533066.33 0.451102211 12 5.413226532 G10:2-1 7755593.333 0.1918531111 12 2.302237333 G10:2-2 1011421 -0.03295263333 12 -0.3954316 G10:2-3 7767009.667 0.1922336556 12 2.306803867 In the table above, the first 1 represents Patient 1 (28319) and the first 2 represents Patient 2 (81362). The second number that immediately follows the dash represents the replicate number. For example, G10:1-1 represents Patient 1 (28319) replicate number 1. PCR Results: Summary Our positive control PCR result was 5.8649688 μg/mL Our negative control PCR result was 0.8283793332 μg/mL Observed results Patient 28319 : Qualitatively, in the pictures of our first patient we clearly saw a bright green color in all three replicates. The three PCR observed product concentrations were 9.646562932 μg/mL, 6.806477868 μg/mL, 5.413226532 μg/mL. Patient 81362 : Qualitatively, in the pictures of our second patient we did not see any green glowing color in any of the three replicates. The three PCR observed product concentrations were 2.302237333 μg/mL, -0.3954316 μg/mL, 2.306803867 μg/mL. Conclusions Patient 28319 : Based off both of our qualitative and quantitative analysis of our results, we can conclude that patient 28319 yields a positive result. This can also be seen by comparing the PCR Product concentrations to our controls values. The values of patient 28319 most closely compared to our positive control value of 5.865 μg/mL. Patient 81362 : Based off both of our qualitative and quantitative analysis of our results, we can conclude that patient 81362 yields a negative result. This can also be seen by comparing the PCR Product concentrations to our controls values. The values of patient 81362 most closely compared to our negative control value of 0.828 μg/mL.