BME100 s2018:Group2 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: Ian Conley
Name: Aisha M Alsuwaidi
Name: Sarah Davis


PCR Reaction Report

While preparing the solutions for the PCR reaction, our group did a good job of staying focused and sharing the responsibilities. The pre-lab readings were helpful, but in the lab, not too practical. The use of the pipettor was very difficult for all members, and we had to try multiple times in some cases to get the liquid into the sample vials. The difference between and the use of the multiple stops was not clear to some members and were a hurdle that was very large. In one case, a member could not get much of the solution out of its original vial and the final reaction did not have the same amount of liquid as all the rest. In some cases, there was residual liquid left in the original vials and may cause some error in the PCR and gel electrophoresis in the next section of the lab. The labeling scheme was consistent with that set up in Lab 4 and was not changed. Overall, practice trials or going over the pre-lab video about how to use the micropipette in class with instructions and details from the professor would have been much more helpful in the long run.

Fluorimeter Procedure

Imaging set-up

  1. Set your camera's timer to three seconds.
  2. Adjust the height of the fluorimeter, so that the drop is the same level as your camera.
  3. Adjust the distance between the slide and your smartphone camera, so that the camera focuses more on the drop. The distance should be >4cm and record the distance.
  4. Put the black box around the fluorimeter and have one flap open. Lower the flap before you take the picture.

Placing Samples onto the Fluorimeter

  1. Place the slide, rough slide down, into the fluorimeter.
  2. Turn on the fluorimeter.
  3. Place the 80 uL SYBR Green 1 solution on the first two dots in the middle of the slide, using the micropipette.
  4. Then place the 80 uL of the PCR or calibration solution onto the drop previously placed.
  5. Make sure that the light is through the drop of solution and adjust the slide as needed for this part.
  6. After imaging, use the micropipette to suck up the solution and adjust the slide to the next set of dots.

Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA

Zero Calf Thymus DNA (H2O calibration) Zero Calf Thymus DNA (H2O Calibration)

Low Calf Thymus DNA (0.5 calibration) Low Calf Thymus DNA (0.5 Calibration)

High Calf Thymus DNA (5 calibration) High Calf Thymus DNA (5 Calibration)

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 13177890 14435223 15559490 14390867.67 1191419.4
2 1 C-2 12300321 12632424 13015413 12649386 357847.6271
1 0.5 C-3 9046300 11059099 12621105 10908834.67 1792133.428
0.5 0.25 C-4 4672426 4753979 5059973 4828792.667 204318.337
0.25 0.125 C-5 7824846 4854867 5218871 5966194.667 1619896.081
0 0 C-6 4387479 4556983 3112914 4019125.333 789365.0214

Calibration curves
Plot 1
Plot 2

Images of Our PCR Negative and Positive Controls



PCR Results: PCR concentrations solved

PCR Product TUBE LABEL MEAN RAWINTDEN PCR Product Concentration (µg /mL) Total Dilution Initial PCR Product Concentration (µg /mL)
+ 12411750 2.1029375 12 25.23525
- 4596242.333 0.149060583 12 1.788727
g1-1 12318643 2.07966075 12 24.955929
g1-2 12725884.33 2.181471083 12 26.177653
g1-3 13998751 2.49968775 12 29.996253
g2-1 3823428 -0.044143 12 -0.529716
g2-2 2455710.667 -0.386072333 12 -4.632868
g2-3 2814304.667 -0.296423833 12 -3.557086

PCR Results: Summary

Control results

  • Our positive control PCR result was 25.23525 μg/mL
  • Our negative control PCR result was 1.788727 μg/mL

Observed results

  • Patient 38066 : Most droplets for patient 1 were showing green glows even before they were put in full darkness, and the images came back with bright green fluorescent colors showing up. The final results were 24.96, 26.18, and 30.00 μg/mL.
  • Patient 66318 : Patient 2's photos were not green and did not show barely any green in the droplet. Any green in the photos could easily be traced back to errors with contamination on the slide itself. Its values were -0.53, -4.63, and -3.56 μg/mL.


  • Patient 38066 : Positive. The values of this patient were consistently close to that of the positive control leading to this conclusion.
  • Patient 66318 : Negative. The patient's values were nowhere near the positive control values, and also were very similar to the negative control.