BME100 s2015:Group1 12pmL4

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Contents

OUR TEAM

Name: Jake PackerRole(s)
Name: Jake Packer
Role(s)
Name: Andre NguyenRole(s)
Name: Andre Nguyen
Role(s)
Name: studentRole(s)
Name: student
Role(s)
Name: Kaylena ConklinRole(s)
Name: Kaylena Conklin
Role(s)
Name: studentRole(s)
Name: student
Role(s)
Name: studentRole(s)
Name: student
Role(s)

LAB 4 WRITE-UP

Protocol

Materials

  • Lab coat and disposable gloves
  • PCR reaction mix, 8 tubes, 50 μL each: Mix contains Taq DNA polymerase, MgCl2, and dNTP's
  • DNA/primer mix, 8 tubes, 50 μL 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 re-use disposable pipette tips or samples will be cross-contaminated
  • Cup for discarded tips
  • Micropipettor
  • OpenPCR machine: shared by two groups


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G1 + Positive control none
G1 - Negative control none
G1 1-1 Patient 1, replicate 1 96837
G1 1-2 Patient 1, replicate 2 96837
G1 1-3 Patient 1, replicate 3 96837
G1 2-1 Patient 2, replicate 1 39617
G1 2-2 Patient 2, replicate 2 39617
G1 2-3 Patient 2, replicate 3 39617


DNA Sample Set-up Procedure

  1. Obtain the DNA samples of the patients along with PCR reaction mix
  2. Label each of the PCR tubes with the desired label of each of the samples that will be tested
  3. Place a new pipette tip onto the micropipettor
  4. Extract 50 μL of PCR reaction mix
  5. Release the PCR mix into the corresponding labeled empty PCR tube
  6. Remove the used pipette tip into a cup
  7. Place a new pipette tip onto the micropipettor
  8. Extract 50 μL of the corresponding patient DNA sample/primer mix
  9. Release patient DNA into the same tube containing the PCR reaction mix
  10. Repeat steps 3-9 for all of the desired samples. Be sure the DNA sample is correctly added to its corresponding PCR tube.
  11. Place the tubes into the thermal cycler. Be sure to wait for another group to place their tubes inside the same thermal cycler.


OpenPCR program

HEATED LID: 100°C

INITIAL STEP: 95°C for 2 minutes

NUMBER OF CYCLES: 35

Denature at 95°C for 30 seconds, Anneal at 57°C for 30 seconds, and Extend at 72°C for 30 seconds

FINAL STEP: 72°C for 2 minutes

FINAL HOLD: 4°C


Bonus Image:PCRBonus.jpg




Research and Development

PCR - The Underlying Technology

Function of components in a PCR reaction. The template DNA is the original copy of base pairs and their specific order. The primers are both the beginning and endpoints of the specific section of the DNA to be copied. Taq polymerase reads single DNA strands and allocates a strand of its base pairs to create a new DNA strand. Deoxyribonucleotides are the base the pairs that taq polymerase attaches to a single strand of DNA to create a new strand.

Thermocycling process. The initial step of 95 degrees celsius for 3 minutes of the thermal cycle is to ensure the DNA is fully "melted" and that it is no longer in its helix shape. The denaturing step in the cycle is the first step in the reoccurring process. The template DNA is split into two separate strands. During the annealing step dNTP's naturally try to bind themselves to the single strand. During the extending step the taq plymerase reads the single stranded DNA and attaches the appropriate dNTP's. The final step allows the DNA to solidify and is the last extension of the cycle. The final hold significantly slows down the reaction and allows for storing.

Base pairs. Adenine and Thymine bind to each other while Cytosine binds to Guanine.

Base pairing thermal cycling. Base pairing occurs during the annealing and extending steps. During the annealing step the base pairs are naturally formed due to 57 degree celsius temperature. During the 72 degree celsius temperature the taq polymerase works at its optimal level attaching base pairs to the single DNA strand.




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