BME100 f2018:Group5 T1030 L5

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Our Team

Name:Rohan Nair
Name:Cesar Diaz
Name: Sydney Gowen
Name: Sumayah Wehelie
Name: Atala Ortiz

Lab 5 Write-Up

PCR Reactions

PART C

  • Lab coat and disposable gloves
  • PCR reaction mix, 8 tubes, 50 microliters each: Mix contains Taq DNA polymerase, MgCl2, dNTP's
  • DNA/primer mix, 8 tubes 50 microliters each: Each mix contains a different template DNA. All tubes have the same forward primer and reverse primer
  • A strip of empty PCR tubes
  • Disposable pipette tips, only use each only once. Never reuse disposable pipette tips. If you do, the samples will become cross-contaminated
  • Cup for discardable tips
  • Micropippettor
  • Open PCR machine: shared by two groups
  1. Cut the PRC tubes in half, leaving two sets of four linked tubes.
  2. using a black marker label the SIDES of the empty tubes with the labels created.
  3. Place tubes back into rack
  4. Begin with the empty tubes labeled as positive control. Utilizing proper pipetting technique, move 50 mirco liters of the PCR reaction mix into the empty tubes. Discard the disposable tip into the collect cup in oder to avoid cross contamination among the patients.
  5. Using a fresh pipette tip, transfer the positive control DNA/primer mix into the same tube. The total volume will be 100 micro liters.
  6. Repeat steps 5 and 6 for the negative control, patients 1 and 2, replicates 1 and 2. Using the proper DNA/primer mix for the appropriate corresponding tubes. After this step is completed all labeled tubes should contain DNA/primer mix plus PCR mix, resulting a 100 mirco liter PRC solution in each tube.
  7. Close lids tightly on the PRC reaction tubes

Procedure

  1. Double check that all of the materials needed are there
    • Buffer solutions: 8 tubes marked with red dots
    • SYBR GREEN 1 solution: 2 tubes labeled with a blue"S"
    • Water @ pH=8: 1 tube labeled H20
    • PCR reaction samples (6 total)
    • Calf Thymus DNA: 5 tubes labeled 0.25, 0.5, 1, 2, and 5
    • Micropipette w/ tips
    • Glass sides w/ hydrophoic coating
    • Temporary pipette tip disposal
    • "Light Box" with assorted parts
    • A Smartphone with functional camera
  2. Label each of the buffer solution tubes to match the PCR reaction sample labels.
  3. Calibrate the pipette to 120 microliters
  4. Transfer each of the PCR reaction samples (100 microliters) from Lab C into its corresponding buffer solution. Make sure to discard the tip of the micropipette before transferring a new solution. Repeat this step until all the PCR sample tubes are empty. .
  5. After transferring the samples, close the lids of the tubes and invert the sample to ensure that the DNA and the buffer solution mix properly.
  6. Set up the fluorescence detection system as demonstrated in lab.
  7. Once set up, calibrate the micropipette to 160 microliters.
  8. Place a 160 microliter drop of water in the middle of the first two rows of the slide using the pipettor so that the drop is pinned and looks like a beach ball.
  9. Turn on excitation light using the switch for the blue LED.
  10. Make sure you camera settings follow the outlined criteria.
    • Flash OFF
    • Set ISO to be greater than or equal to 800
    • Set white balance to auto
    • Set exposure and saturation to highest setting
    • Set contrast to lowest setting
  11. Place smartphone perpendicular to slide in the fluorescence detection system using a cradle. Be sure to adjust the height of the fluorimeter using plastic trays to make the smartphone camera roughly parallel with slide.
  12. Adjust the camera's distance from the system so that it is as close to the slide as possible with out creating a blurry image.
  13. Take at least three images of the 160 microliter drop of water when the system is closed.
  14. Calibrate the micropipette to 80 microliters.
  15. Take an 80 microliter solution of SYBER GREEN 1 and place it in the middle of the first two rows of the slide using the pipettor. Add an 80 microliter solution of one of calf thymus solutions listed as follows: 0, 0.25, 0.5, 1, 2, and 5. This
  16. Align the drop with the blue LED
  17. Use the timer on your camera to take a picture of the drop when the system is closed.
  18. Again, take at least three images drop of water when the system is closed.
  19. Remove the box. Try not to move your smartphone.
  20. Use the pipettor to remove the solution from surface of the slide.
  21. Put in a fresh slide.
  22. Repeat steps 14-21 for the other concentrations of calf thymus DNA.
  23. Clean up work station

Data Collection and Analysis

PART D

Smartphone 1: Model Samsung Galaxy S8
Optional Smartphone 2: Model N/A
  • Disclaimer: One data sample was lost due to technical difficulties (The pipette broke the tube. Too much force applied.)

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