BME100 s2018:Group4 W1030 L5

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BME 100 Spring 2018 Home
Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
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Name: Amanda Tran
Name: Maren Eltze
Name: Jonathan Kendall-Jackson
Name: Ceasar Carreto


PCR Reaction Report

Our group was able to successfully use the micro-pipettes to carry out the PCR reaction. Almost every member of our group had used a micro-pipette before, so their incorporation into the lab did not prove to be an especially difficult challenge. Despite having background knowledge and experience, the pre-lab reading and simulation helped us review the concepts and steps behind using a micro-pipette, and thus the pre-lab was very useful. We correctly used the pipette throughout the experiment, pressing the plunger down to the first stop when taking up the liquid and pressing the plunger down to the second stop when releasing it. In our experiment, every one of our final reactions looked like they had exactly the same amount of liquid (as expected). It is possible that there was a very slight discrepancy in size between some of the reactions, perhaps due to small bubbles, but we were careful to be as consistent with all of the liquid sample sizes as possible. There was no easily visible liquid left at the bottom of the tubes containing DNA/PCR reaction mix, however there is still a possibility that there was a very small amount remaining due to human error. We did not change our labeling scheme; it was kept consistent with that of Lab 4.

Fluorimeter Procedure

Imaging set-up

The primary objective of this section of the lab is to use fluorescence to detect DNA using a smartphone. Settings on the camera were changed in addition to the download of the computer software Image J. The materials collected were a tray of sample tubes, a micropipette, pipette tips, our patient PCR reaction tubes, glass slides, and a Fluorimeter kit, which included a fluorimeter, phone stand, and black box.

Placing Samples onto the Fluorimeter

  1. Place the slide into the fluorimeter with the rough side facing down
  2. Place smartphone on its stand and adjust the fluorimeter and stand until a close and in focus picture of the droplet can be taken
  3. Pipette 80 µL of SYBR GREEN 1 in between the two rows on the slide
  4. Pipette 80 µL of one of the Calf Thymus or PCR solutions onto the SYBR GREEN 1 drop on the slide
  5. Align the drop and LED light by moving the slide on which the drop rests
  6. Place box over the fluorimeter, with one flap open
  7. Make sure the camera is still focused on the drop, then start the camera timer and close the flap
  8. After the picture is taken, remove the box and remove the 160 µL drop using the micropipette
  9. Discard the liquid waste into the hazardous waste container
  10. Repeat these steps, but change where on the slide the calf thymus solution or PCR is placed (until 5 possible measurement positions are used) ensuring that both the PCR and Calf Thymus samples have been imaged in all of the possible positions

Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA

Image J for 5μg/mL sample:

Image J for 0.5μg/mL sample:

Image J for zero DNA 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 MEAN Standard Deviation
Image 1 Image 2 Image 3
5 2.5 C-1 4185234 5365929 6015000 5188721 927665.3007
2 1 C-2 3743365 3580604 3746131 3690033.33 94778.6734
1 0.5 C-3 2766824 2549043 2487094 2600987 146921.246
0.5 0.25 C-4 1009030 986727 872285 956014 73363.922
0.25 0.125 C-5 779044 830556 859594 823064.667 40794.187
0 0 C-6 175705 166114 563156 301658.333 226514.3904

Calibration curves

Images of Our PCR Negative and Positive Controls

Positive Control - Image J:

Negative Control - Image J:

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)
G4 + 4224095.7 3.236 12 38.832
G4 - -438702 -0.8554 12 -10.2648
G4 1-1 9446639 7.817 12 93.804
G4 1-2 9341033 7.725 12 92.7
G4 1-3 8437615.3 6.932 12 83.184
G4 2-1 -384931 -0.807 12 -9.684
G4 2-2 -334290.33 -0.762 12 -9.144
G4 2-3 -1553485 -1.831 12 -21.972

PCR Results: Summary

  • Our positive control PCR result was 38.832 μg/mL
  • Our negative control PCR result was -10.26 μg/mL

Observed results

  • Patient 38270 : The photos from patient 38270 were observed to have a more green appearance, and when observed in Image J in the green channel, they are very light. Additionally, the quantitative results for this patient were 93.804 μg/mL, 92.7 μg/mL, and 83.184 μg/mL.
  • Patient 31977 : The photos from patient 31977 were observed to not omit any fluorescence under the fluorimeter, and when observed in Image J in the green channel, the images are very dark. The quantitative results for this patient were -9.684 μg/mL, -9.144 μg/mL, and -21.972 μg/mL.


  • Patient 38270 : The positive control result was 38.832 μg/mL, which was comparable to the results of Patient 38270. Therefore, it can be concluded that Patient 38270 is positive for the Disease SNP.
  • Patient 31977 : The negative control result was -10.26 μg/mL, which clearly parallels to the results observed from Patient 31977. Therefore, it can be concluded that Patient 31977 is negative for the Disease SNP.