BME100 s2018:Group1 W1030 L5

From OpenWetWare
Jump to navigationJump to search
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
Course Logistics For Instructors
Wiki Editing Help


Name: Madison Ryder
Name: Georgia Sullivan
Name: Erik Halsband


PCR Reaction Report

Our team was successfully able to carry out the PCR reaction. We had previous experience working with micro-pipettes, so we were good at differentiating between the first stop and the second stop. The first stop was used to suck up liquid, and the second stop was used to dispel it. The final reactions all had 100 micro-liters of liquid, and all the tubes looked like they contained the same size sample. They should have all had the same amount of liquid, because we were careful not to get bubbles when pipetting the samples. The pre-lab reading helped to clarify the order of the steps, meaning whether or not the reaction mix or the sample should be added first. There did not appear to be liquid left in the sample tubes, but there may have been some small drops due to human error. Our labeling scheme was successful because our labels were only a few characters long, and the tubes did not have much room to write more on them.

Fluorimeter Procedure

Imaging set-up
First the glass slide was placed into the fluorimeter with the smooth side down, and it was placed onto a plastic box to raise it up higher. Then the blue LED light was turned on, and 160 micro-liters of water was placed onto the glass slide in between the first and second row of the light. The smartphone cradle was placed in front of the fluorimeter, and the smartphone was placed so that it viewed the water droplet from the side, parallel to the LED light. The distance between the smartphone and the drop was adjusted until the smartphone could properly adjust to the drop, and this distance was recorded. The camera was adjusted, and a timer was set for 10 seconds. After starting the camera timer, the box was placed over the fluorimeter smartphone system to keep as much light as possible out of the picture. To set up the camera, turn flash off, set ISO to 800 if possible, increase exposure and saturation to the highest level, set white balance to auto, and decrease the contrast.

Placing Samples onto the Fluorimeter

  1. With gloves, place glass slide, smooth side down, on fluorimeter, and turn it on
  2. Place smartphone in cradle, and place it to the edge of the fluorimeter, parallel to the LED light
  3. Adjust height of fluorimeter until the camera views the glass slide from the side, instead of from above
  4. Focus camera, change camera settings, and measure distance between LED light and smartphone camera
  5. Place disposable top on micro-pipettor
  6. Push to first stop and suction 80 micro-liters of SYBR green 1 solution by letting go of first stop, and place onto middle row between LED light by pushing to second stop
  7. Dispose tip, replace with a new tip
  8. Push to first stop and suction 80 micro-liters of one of the calf thymus solutions and place on top of SYBR green 1 drop by pushing to second stop
  9. Focus camera on drop and place box over the fluorimeter, with one flap open
  10. Start timer, and close flap while pictures are being taken
  11. Remove box and suction off of the slide after taking the pictures by pressing to first stop and releasing
  12. Dispose of liquid in disposable waste cup
  13. Repeat steps 1-6 with the rest of the calf thymus solutions, but change which part of the glass slide the drop is placed onto by putting a new slide onto the fluorimeter
  14. Now all the steps should be repeated, but instead of using the calf thymus solutions, 80 micro-liters of each of the PCR reaction samples should be used

Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA

Zero Calf Thymus DNA (0 μg/mL sample):

Low Calf Thymus DNA (0.5 μg/mL sample):

High Calf Thymus DNA (5 μg/mL sample):

Calibrator Mean Values

Sample Number Image number Final DNA concentration in SYBR Green I solution (µg/mL) AREA Mean Pixel Value RAWINTDEN OF THE DROP RAW INTDEN OF THE BACKGROUND RAWINTDEN Drop - Background
C-1 1 5 265185 192.93 51162118 2079796 49082322
C-1 2 5 265684 193.348 51369528 2237676 49131852
C-1 3 5 267113 207.092 55317001 11499459 43817542
C-2 1 2 237396 184.617 43827289 2089020 41738269
C-2 2 2 243712 183.984 44839138 2042618 42796520
C-2 3 2 244606 184.082 45027596 2152298 42875298
C-3 1 1 254228 154.364 39243554 2373685 36869869
C-3 2 1 257662 153.317 39503843 2418686 37085157
C-3 3 1 254932 153.579 39152121 2411159 36740962
C-4 1 0.5 273198 143.886 39309395 2614852 36694543
C-4 2 0.5 257662 146.021 37624111 2503272 35120839
C-4 3 0.5 255443 146.425 37403131 2397136 35005995
C-5 1 0.25 246732 142.616 35187891 2356299 32831592
C-5 2 0.25 254748 140.816 35872494 2489384 33383110
C-5 3 0.25 256516 140.19 35960905 2479223 33481682
C-6 1 0 233720 123.19 28791903 2371237 26420666
C-6 2 0 248808 121.504 30231276 2557887 27673389
C-6 3 0 243484 122.824 29905650 2440516 27465134

Calibration curves

Images of Our PCR Negative and Positive Controls

Negative control:

Positive control:

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)
+C 46112768 5.370922667 12 64.451072
-C 30604246 0.201415333 12 2.416984
1-1 8181594 -7.272802 12 -87.273624
1-2 23597367 -2.134211 12 -25.610532
1-3 22359718 -2.546760667 12 -30.561128
2-1 16752175 -4.415941667 12 -52.9913
2-2 11782053 -6.072649 12 -72.871788
2-3 19763704 -3.412098667 12 -40.945184

PCR Results: Summary

  • Our positive control PCR result was 64.45 μg/mL
  • Our negative control PCR result was 2.42 μg/mL

Observed results

  • Patient 29486: -47.81 μg/mL

To the eye, the images for this patient looked brighter and closer to the positive control than the images for the other patient. These images have a greener tint.

  • Patient 76146: -55.60 μg/mL

In comparison, these images are more dim and blue tinted.


  • Patient 29486: Positive
  • Patient 76146: Negative

For both patients, the data was too inconsistent to provide a definitive positive or negative reading. All of the concentrations came back with negative values, which is theoretically impossible. Errors were made either with the PCR reaction, the fluorimeter, or the imageJ analysis. However, to the naked eye, the first patient's images were brighter and more similar to the positive control than the second patient's, leading to the conclusion that the first patient is positive and the second patient is negative. However, in order to confirm these conclusions further testing must be done.