BME100 f2015:Group11 8amL5

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OUR TEAM

Name: Maria Roman
Name: Teddy Valenzuela
Name: Kyle Greenzweig
Name: Dean Spyres
Name: Ryan Tenorio
Name: Kyra Temple


LAB 5 WRITE-UP

PCR Reaction Report

Before attending lab, we read the pre-lab reading and completed the quiz. This prepareed us for pipetting the DNA samples and the PCR reaction mix. Each DNA sample, as well as the positive and negative control were added to each labelled PCR reaction tube. We labelled the tubes on the front side using a sharpie: P (positive), N (negative), 1 (patient 1), 2 (patient 2). We pipetted, drawing the liquid up using the first stop, and pushed the liquid out using the second stop. A new pipette tip was used for each sample. After they were all filled, we placed them in the OpenPCR.

Fluorimeter Procedure

Smart Phone Camera Settings

  • Type of Smartphone: iPhone 6
    • Flash: none
    • ISO setting:2000
    • White Balance: N/A
    • Exposure:f/2.2
    • Saturation:
    • Contrast:


Camera set-up
Place the smartphone on a cradle. Then move it to a position so that its camera can see the slide from the edge on. Adjust height of fluorimeter if needed and make sure the camera's timer is set to 3 seconds. Once ready to take a picture of the drop on the slide, adjust the distance between the smartphone and the fluorimeter so that the camera is focused on the drop.

  • Distance between the smart phone cradle and drop = 6 cm


Placing Samples onto the Fluorimeter

  1. Make sure the fluorimeter is on, the camera is set, and the smooth side of the slide is facing down. Place an 80 microliter drop of the SYBR Green I solution in the middle of the slide on the first two clear circles.
  2. Take an 80 microliter drop of the sample and place it on top of the SYBR Green I Solution. Make sure the light illuminates the drop's center.
  3. Cover the fluorimeter with the lightbox while leaving a flap up, make sure the camera is focused, press the timer button on the camera, and lower the flap.
  4. Take the drop from the slide and place it in the liquid waste container. Move the slide so that the light is in the center of the next two circles.
  5. Repeat these steps with all five positions on the slide and place slide in SHARPS container once done.


Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA

5 μg/mL Sample Kyle5.jpeg

0.5 μg/mL Sample Kyle3.jpeg

0 μg/mL Sample Kyle1.jpeg

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
5 2.5 C-1 10937732 10937732 10937732 10937732 0
2 1 C-2 10479042 10479042 10479042 10479042 0
1 0.5 C-3 7564693 7564693 7564693 7564693 0
0.5 0.25 C-4 6349244 6349244 6349244 6349244 0
0.25 0.125 C-5 4797928 4797928 4797928 4797928 0
0 0 C-6 4101748 4101748 4101748 4101748 0


Calibration curves

Group118ampic1.jpg


Images of Our PCR Negative and Positive Controls

Positive Control Group118amPositive.jpg

Negative Control Group118amNegative.jpeg

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)
G11 + 14058421.3 3.3528071 12 40.2336852
G11- 2672211.67 -0.44259611 12 -5.31115332
G11 1-1 11292665 2.430888333 12 29.17066
G11 1-2 11678217 2.559405667 12 30.712868
G11 1-3 13581887 3.193962333 12 38.327548
G11 2-1 2827443 -0.390852333 12 -4.690227996
G11 2-2 3239641 -0.253453 12 -3.041436
G11 2-3 2486281 -0.504573 12 -6.054876


PCR Results: Summary

  • Our positive control PCR result was 40.2336852 μg/mL
  • Our negative control PCR result was -5.31115332 μg/mL


Observed results

  • Patient 24467 : The images that were taken of this patients DNA were fluorescent green. It was fairly easy to observe the green color within the drop. The quantitative results are 29.17066, 30.712868, and 38.327548 μg/mL for product tubes 1-1, 1-2, and 1-3 respectively.
  • Patient 87309 : There was no visible color on the images taken of this patients DNA. The quantitative results are -4.690227996, -3.041436, and -6.054876 μg/mL for the product tubes 2-1, 2-2, and 2-3 respectively.


Conclusions

  • Patient 24467 : In conclusion, patient 1 is positive due to the observed color and the overall product concentration values being very close to that of the positive thresh hold and very far away from the negative thresh hold.
  • Patient 87309 :In conclusion, patient 2 is negative due to the lack of observable color and the overall product concentration levels being very close to that of the negative thresh hold and very far away from the positive thresh hold.