BME100 f2016:Group6 W8AM L5

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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
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OUR TEAM

Andee Wallace Peplowski
Charlotte Burke
Suleica Garcia
Sarah Unger
Astha Chourasia


LAB 5 WRITE-UP

PCR Reaction Report

The lab group first micropipetted 50 microliters (µL) of each individual DNA/primer mix into properly labeled tubes. Taking care not to cross contaminate each sample mix, another 50 µL of PCR reaction mix was added and mixed with the DNA/primer solutions. Pipette tips were carefully disposed before each individual solution. During the first three mixes, the micropipetter was new to the technique and could have been a source of error for measurement.

These tubes were then placed in the PCR machine and run through 25 cycles. The lid was set to 100°C. The initial step was set to 95°C for two minutes. The cycles were set for 30 seconds each to denature at 95°C, anneal at 57°C and extend at 72°C. The computer connected to the PCR machine logged out the user after 10 minutes, and although this did not appear to affect the machine it could have caused error or a glitch in the cycles.

A few members in our group have had experience with pipetting so they were able to help everyone during the lab. The pre-lab helped because the simulation for the pre-lab was very clear and detailed enough to help understand what we would be doing in the lab. We understood the difference between the first and second stop on the pipettor very easily because of the pre-lab simulation. We used it as guidance during the lab. The final reaction did not have the same amount of liquid due to air bubbles in the solution and transfer process, most likely caused by human error. Additionally, there was not any excess liquid left in the tubes that the DNA samples and PCR reaction were contained in. We did not have to adjust or change the labeling system that we established in the previous lab.

Fluorimeter Procedure

Imaging set-up
To properly capture images from the fluorimeter, a smartphone camera needs to be set up. To make sure the Camera is at the same level of the beam, try adding support underneath the device to raise it. Click on the timer on the camera app and set the timer to at least 3 seconds. Capture each image after lowering down the end flap to eliminate any exterior light coming through.



Placing Samples onto the Fluorimeter

  1. [Step one: Find the smooth side of the slide. ]
  2. [Step two:Turn on the Fluorimeter and place the slide in the device smooth side down. ]
  3. [Step three: Set up the camera with a 3-second timer on the cradle and adjust the height of the device to make sure the droplet is in perfect view.]
  4. [Step Four: Place 80 uL drop of SYBR green solution in the middle of the slide towards the top edge (first two bubbles). ]
  5. [Step Five: Place 80 uL drop of Calibration sample on top of the SYBR green solution. Make sure the slide is adjusted so the droplet is in the center of the beam. ]
  6. [Step Six: Record the distance of the Camera from the Device. Cover the Device with the black box, with one flap open for easy access. ]
  7. [Step Seven: Take a picture and close the end flap before the shutter takes the snapshots. ]
  8. [Step Eight: After taking 3 different pictures, remove the 160 uL solution and dispose in the liquid disposal. Move the slide up to the next row of circles. ]
  9. [Step Nine: Repeat the above steps until you have used all 5 measurement positions on the slide. ]




Data Collection and Analysis

High Calf Thymus DNA (5μ/mL) Image of High Calf Thymus DNA

Low Calf Thymus DNA (0.5μ/mL) Image of Low Calf Thymus DNA

Zero Calf Thymus DNA (0μ/mL) Image of Zero Calf Thymus


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 2.5 5728 165.1 945693 17111 928582
C-1 2 2.5 5054 166.689 842447 19233 823214
C-1 3 2.5 5586 165.741 925830 21179 904651
C-2 1 1 5646 137.762 777807 30685 747122
C-2 2 1 14195 145.592 2066676 131606 1935070
C-2 3 1 14130 158.958 2246077 74551 2171526
C-3 1 0.5 5206 155.018 807026 23160 783866
C-3 2 0.5 5206 145.66 758305 36886 721419
C-3 3 0.5 5689 140.589 799810 46994 752816
C-4 1 0.25 8960 129.482 1160158 99554 1060604
C-4 2 0.25 8079 135.419 1094049 81740 1012309
C-4 3 0.25 23044 120.623 2779640 215749 2563891
C-5 1 0.125 9260 117.697 1089877 102904 986973
C-5 2 0.125 22062 117.001 2581284 228167 2353117
C-5 3 0.125 20962 120.203 2519702 217999 2301703
C-6 1 0 7240 82.913 600292 26087 574205
C-6 2 0 7317 76.106 556867 24329 532538
C-6 3 0 6908 69.069 477128 41917 435211

Calibration curves
Screenshot 2016-11-08 23.00.56.png

Screenshot 2016-11-08 23.01.43.png

Images of Our PCR Negative and Positive Controls Posscon.png Image of Positive Control

Neggcon.png Image of Negative Control

PCR Results: PCR concentrations solved

PCR Product Concentration (µg /mL) Total Dilution "Initial PCR Product Concentration
(µg /mL)
4.42696942 12 53.12363304
0.667358087 12 8.008297048
-0.030511217 12 -0.366134599
0.006802175 12 0.081626101
0.347821398 12 4.173856773
0.311751309 12 3.741015711
0.259756807 12 3.117081686
2.096657604 12 25.15989125


PCR Results: Summary

  • Our positive control PCR result was 53.120 μg/mL
  • Our negative control PCR result was 8.010 μg/mL


Observed results

  • Patient 27468: After observing the droplet under the ImageJ software, we noticed a strong appearance of green towards the center of the drop. 4.17 μg/mL was observed for Patient 27468.
  • Patient 96944: While observing the droplet during the experiment, The drop for Patient 96944 showed no difference in appearance (naked eye observation) as compared to the Patient 27468 droplet. 25.16 μg/mL was observed for Patient 96944.


Conclusions

  • Patient 27468: Negative___: We concluded the patient was negative based on the values that matched our negative control.
  • Patient 96944:Negative___: We concluded the patient was negative based on the values that matched our negative control.