BME100 f2015:Group7 8amL5

From OpenWetWare
Jump to: navigation, search
Owwnotebook icon.png BME 100 Fall 2015 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
BME494 Asu logo.png

OUR TEAM

Name: Tristan Loveday
Name: Jeremy Atkinson
Name: Kelly Harper
Name: Andrew Medina
Name: Farah Alyasari
Name: Rafael Lopez


LAB 5 WRITE-UP

PCR Reaction Report

The pipetting went fairly smoothly because we took our time to make sure all the samples were transferred properly. We made sure that we understood the difference between the first and second stop on the pipettor before we started pipetting, and there was no visible difference between the different PCR reaction tubes after we were finished. While we were pipetting, we noticed some water on the sides of the tubes with samples and master mix; we tried to use the pipette tip to move these water bubbles down to the rest of the liquid, with which we were fairly successful. At the end, there was a small amount of liquid left in a couple of the master mix/sample tubes; however, the amount of liquid was very small and hopefully had no impact on our reactions. We kept our labeling scheme the same, as our labels fit on the PCR tubes.

Fluorimeter Procedure

Smart Phone Camera Settings

  • Type of Smartphone:Galaxy Note 5
    • Flash:off
    • ISO setting:800
    • White Balance: auto
    • Exposure:highest
    • Saturation:highest
    • Contrast:lowest


Camera set-up

Example328746598723.jpg

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


Placing Samples onto the Fluorimeter

  1. Place a clean slide onto the stand, with the beam of light going across the slide between the first and second row of dots
  2. Using the micropipeter, place 80 µL of CYBR GREEN 1 between the first and second row of dots
  3. Repeat step 2 using 80 µL of the sample, creating a droplet of 160 µL
  4. set up the smartphone and cradle at 6 cm from the droplet
  5. enclose the smartphone cradle and stand in the lightbox
  6. Take a focused picture of the droplet, ensuring that the lightbox is closed to eliminate excess light
  7. repeat step 6 for a total of 3 pictures of the droplet
  8. Remove the droplet using the micropipeter and dispose of into a liquid contamination container
  9. Repeat steps 2-8 between two rows of the slide that have not been used, using a new slide if the current slide has no remaining sections



Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA


High: High94687Thymus.jpg Low: Low9687Thymus.jpg Zero: Zero94687Thymus.jpg Calibrator Mean Values


Image No. Initial Concentration of 2X Calf Thymus DNA solution (µg/mL) Volume of the 2X DNA solution (µL) Volume of the SYBR GREEN 1 Dye solution (µL) Final DNA concentration in SYBR Green 1 solution (µg/mL) Area Mean Pixel Value RAW INTDEN of the drop RAW INTDEN of background drop-background mean stdev
1 0 80 80 0 94092 58.537 5507906 893875 4614031 3846014 1155168
2 0 80 80 0 94092 54.743 5150917 744439 4406478 3846014 1155168
3 0 80 80 0 94092 36.512 3435490 917955 2517535 3846014 1155168
1 0.25 80 80 0.125 94092 40.136 3776479 914123 2862356 8419486 5612025
2 0.25 80 80 0.125 94092 96.217 9053251 741990 8311261 8419486 5612025
3 0.25 80 80 0.125 94092 153.958 14486231 401389 14084842 8419486 5612025
1 0.5 80 80 0.25 94092 154.642 14548881 406768 14142113 13686515 740442
2 0.5 80 80 0.25 94092 155.801 14659636 574360 14085276 13686515 740442
3 0.5 80 80 0.25 94092 141.559 13319604 487448 12832156 13686515 740442
1 1 80 80 0.5 94092 150.074 14120798 578236 13542526 16556408 2620299
2 1 80 80 0.5 94092 195.209 18367568 72899 18294669 16556408 2620299
3 1 80 80 0.5 94092 192.043 18069699 237704 17831995 16556408 2620299
1 2 80 80 1 94092 175.337 16497850 140985 16356865 16302758 79202
2 2 80 80 1 94092 176.31 16589390 249831 16339559 16302758 79202
3 2 80 80 1 94092 174.256 16396106 184256 16211850 16302758 79202
1 5 80 80 2.5 94092 189.254 17834387 135885 17698502 17674084 127357
2 5 80 80 2.5 94092 188.669 17752211 215925 17536286 17674084 127357
3 5 80 80 2.5 94092 190.491 17923659 136194 17787465 17674084 127357


Calibration curves

Calibration13643245.jpg Calibration2234245345.jpg

Images of Our PCR Negative and Positive Controls

Negative: Negative846115control.jpg Positive: Positive846115control.jpg

PCR Results: PCR concentrations solved

Image No. Sample No. Area Mean Pixel Value RAW INTDEN of the drop RAW INTDEN of background drop-background mean stdev
1 7- + 94092 112.907 10623662 162252 10461410 9845694 1152910
2 7- + 94092 113.917 10718641 158615 10560026 9845694 1152910
3 7- + 94092 96.166 9048494 532848 8515646 9845694 1152910
1 7-1.1 94092 98.454 9263719 527349 8736370 8437331 639508
2 7-1.1 94092 87.533 8238034 534946 7703088 8437331 639508
3 7-1.1 94092 97.762 9198625 326088 8872537 8437331 639508
1 7-1.2 94092 96.725 9101032 197410 8903622 9567110 577777
2 7-1.2 94092 110.965 10440939 481558 9959381 9567110 577777
3 7-1.2 94092 113.219 10652991 814662 9838329 9567110 577777
1 7-1.3 94092 108.587 10217171 9909718 307453 6367965 5252061
2 7-1.3 94092 109.509 10303910 713864 9590046 6367965 5252061
3 7-1.3 94092 105.479 9924751 718355 9206396 6367965 5252061
1 7- - 94092 106.645 10034421 718304 9316117 8809274 446639
2 7- - 94092 96.731 9101598 628328 8473270 8809274 446639
3 7- - 94092 99.797 9390065 751628 8638437 8809274 446639
1 7-2.1 94092 100.663 9471557 750034 8721523 7372011 1181741
2 7-2.1 94092 76.491 7197149 674898 6522251 7372011 1181741
3 7-2.1 94092 80.137 7540242 667983 6872259 7372011 1181741
1 7-2.2 94092 80.575 7581486 664167 6917319 7930410 3996626
2 7-2.2 94092 133.004 12514647 178556 12336091 7930410 3996626
3 7-2.2 94092 55.939 5263418 725598 4537820 7930410 3996626
1 7-2.3 94092 199.475 18768976 302792 18466184 12224300 5407461
2 7-2.3 94092 107.828 10145741 901673 9244068 12224300 5407461
3 7-2.3 94092 103.181 9708498 745850 8962648 12224300 5407461


PCR Results: Summary

  • Our positive control PCR result was 3.691388 μg/mL
  • Our negative control PCR result was 1.618549334 μg/mL


Observed results

  • Patient 1 (54502) : 1-1: 0.874663334 μg/mL 1-2: 3.134221334 μg/mL 1-3: -3.26407 μg/mL

The samples for patient 1 appeared very clear. It is impossible to distinguish any green color by simply looking at the images.

  • Patient 2 (84174) : 2-1: -1.255978 μg/mL 2-2: -0.13918 μg/mL 2-3: 8.4486 μg/mL

The samples from 2-1 and 2-2 are both clear. However, the samples from 2-3 appear green.

Conclusions

  • Patient 1 (54502) : All the pictures from patient 1 appeared clear, and two of the samples came out below the negative control value. Every sample was below the positive control value. For these reasons, the patient 1 sample failed to replicate during PCR and is therefore negative.
  • Patient 2 (84174) : Our data for patient 2 appears to be flawed based on the fact that two of the samples came out below the negative control, while one came out far above the positive control. This correlates with the visual appearance of our pictures. Because sample 2-3 had such a high DNA concentration value, it appears to be an outlier that may be the result of contamination. Based on samples 2-1 and 2-2, patient 2's DNA also failed to replicate, and is therefore negative.