BME100 f2015:Group4 8amL5

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OUR TEAM

Name: Anastasia Wyatt
Name: Austen Bennett
Name: Bhavna Ramesh
Name: Michael Vietz
Name: Kendra Starkel
Name: Drew Higgins


LAB 5 WRITE-UP

PCR Reaction Report

The pipetting tutorials were very helpful for the completion of this lab exercise. Our group was able to come into the lab with an underlying knowledge of micropipetting because of the pre lab activities. Due to the pre-lab exercises we easily understood the difference between the first and second stop on the pipettor. The first stop was used before placing the pipettor into the liquid, which was then slowly released in order to bring the liquid into the pipettor. The second stop was used to deposit the liquid into our sample tubes. However, we didn't have the same amount of liquid in each of our final reactions and there were varying small amounts of leftover liquid in our tubes containing the DNA samples and PCR reaction mix. Our labeling scheme was effective and helped us to identify which PCR reactions were ours.

Fluorimeter Procedure

Smart Phone Camera Settings

  • Type of Smartphone: iPhone 5
    • Flash: off
    • ISO setting: 800
    • White Balance: N/A
    • Exposure: N/A
    • Saturation: N/A
    • Contrast: N/A

Camera set-up
Place the phone into the cradle vertically, make sure the phone isn't slanted, adjust the height of the fluorimeter in order to obtain the best image of the drop on the phone. The drop should be focused on the top of the image. In order to focus the image the screen of the phone can be tapped on where the focus point is.

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

Placing Samples onto the Fluorimeter

  1. As a method of calibration of the fluorimeter, take 160 μL of water using the micropipettor and place it in the middle column of your glass slide in the shape of a drop so that the the drop covers the first two circles in that column.
  2. Then, turn on the blue LED light and adjust if needed such that the light passes through the center of the drop.
  3. Next, set up your camera in the cradle the necessary distance from the fluorimeter such that the camera is focused on the drop of water and is getting a nearly-perfect side view. Be sure to record this distance to ensure consistency for the rest of your lab.
  4. Suck up the water drop with your micropipettor and discard it in your liquid waste cup.
  5. Now, take 80 μL of the SYBR Green I solution and place it on the second two circles in the middle row of your slide.
  6. On the same drop, add 80 μL of the first concentration of the calf thymus solution.
  7. Adjust your LED light so that it is passing through the center of the drop.
  8. Focus your camera and press the capture button, quickly closing the lid of the light box within that three seconds to ensure good picture quality.
  9. Take two more images of the same sample drop to make sure your picture is focused.
  10. Take the light box off the fluorimeter without moving the cradle with the smartphone.
  11. Remove the drop from the slide with the micropipettor and dispose of it in the liquid waste cup.
  12. Repeat steps 5-11 with the remaining concentrations of the calf thymus solution.
  13. This exact same procedure will be repeated with the PCR reaction samples of your group. In order to create the solutions that will be tested, transfer 100 μL from your PCR tubes into the respective matching buffer tubes."
  14. After you mix the buffer with your DNA, the solutions are ready to be placed on the fluorimeter and analyzed. Perform steps 5-11.


Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA

5 μg/mL sample
BME5permlGroup4.PNG

.5 μg/mL sample
BME.5permlGroup4.jpg

0 μg/mL sample
BME0permlGroup4.jpg

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 358894 345832 322802 342509.3333 18273.97607
2 1 C-2 281067 266773 305216 284352 19430.88987
1 0.5 C-3 232134 240197 250435 240922 9172.015536
0.5 0.25 C-4 252572 317224 326227 298674.3333 40178.75504
0.25 0.125 C-5 205264 208678 169573 194505 21659.11626
0 0 C-6 215754 236358 275949 242687 30592.51031


Calibration curves

Dotplot1.jpg
Dotplot2.jpg

Images of Our PCR Negative and Positive Controls

Negative Control
BMEnegativeGroup4.jpg

Positive Control
BMEpositveGroup4.jpg

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)
Positive 27776150.33 1267.153289 12 15205.83946
Negative 14521618.67 657.3062381 12 7887.674857
1,1 11259218.67 507.2017253 12 6086.420704
1,2 12652064.67 571.2872054 12 6855.446465
1,3 11927094 537.9309742 12 6455.17169
2,1 16967863 769.8590562 12 9238.308674
2,2 23174880.33 1055.446756 12 12665.36107
2,3 22622418 1030.027712 12 12360.33255


PCR Results: Summary

  • Our positive control PCR result was 15205.83946 μg/mL
  • Our negative control PCR result was 7887.674857 μg/mL


Observed results

  • Patient 86057 : The images of this patient were dark with very little if not any luminescence. They showed very little green light compared to the positive droplets. The observed concentration for this patient had an average of 6465.6796 μg/mL.
  • Patient 31866 : The images of this patient showed more green light, which was very similar to the positive droplets. Rather than a mostly black image, these images showed luminescence inside of them. The observed concentration of the droplets from this patient had an average of 11421.334 μg/mL.


Conclusions

  • Patient 86057 : When looking at the qualitative data, this patient appears to be similar to the negative control due to the lack of green light within the droplets. Also, the average concentration of 6465.6796 μg/mL was very close to the concentration of the negative sample which was 7887.674857 μg/mL. Due to these factors it was concluded that this patient was negative.
  • Patient 31866 : When looking at the qualitative data for this patient, the droplets are similar to the positive control images which contain more green light throughout the droplets. Also, the average concentration of 11421.334 μg/mL was very close to the concentration of the positive sample which was 15205.83946 μg/mL. Due to these factors it was concluded that this patient was positive.