BME100 f2014:Group23 L4

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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
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OUR TEAM

Name: Danielle Beach
Name: Cesar Marin
Name: Kassandra Flores
Name: Brady Dennison
Name: Joshua Kahn
Name: Ted Kyriacou

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
G23 + Positive control none
G23 - Negative control none
G23 1-1 Patient 1, replicate 1 33849
G23 1-2 Patient 1, replicate 2 33849
G23 1-3 Patient 1, replicate 3 33849
G23 2-1 Patient 2, replicate 1 19961
G23 2-2 Patient 2, replicate 2 19961
G23 2-3 Patient 2, replicate 3 19961


DNA Sample Set-up Procedure

  1. Take the 6 samples of DNA above, and divide them into two separate group and label each of the tubes according to which patient and which sample is contained in the tube.
  2. Create another two PCR tubes, and label them as the positive and negative controls
  3. Calibrate a micro pipet to 50μL
  4. Transfer 50μL of PCR mix into each of the 8 PCR tubes. The mix contains the DNA polymerase as well as the primers.
  5. Transfer each of the patients DNA solution into the PCR tubes.
  6. Make sure the tubes are organized to avoid confusion, and mistakenly transfer the wrong DNA into the wrong tube.
  7. Make sure to dispose of Micro Pipet tips after each transfer to avoid contamination.
  8. At the end, there should be 8 PCR tubes containing 100μL each, 50μL of the DNA and 50μL of the PCR mix.
  9. Place all 8 PCR tubes into thermo cycler.
  10. Using the Thermo Cycler program, start the PCR process of heating and cooling.

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

The OpenPCR program is contained in the computer in the thermo cycler. The information above contains all the data that is inputed into the system. The process begins when the temperature is raised to all the DNA to denature and to split apart into to separate strands. This is the Initial Step, heating the solution to 95 degrees celsius. Then, the DNA is cooled, and the primers added to the solution attach to their binding sites on the DNA, this it the "Anneal" portion described above, at 57 degrees celsius. After this, taq DNA polymerase begins to add nucleotides to the DNA, replicating it. This process is in the "Extend" portion in the data above, at 72 degrees celsius. This is then repeated in a certain amount of cycles to exponentially increase the amount of replicated DNA of that specific portion selected by the primers. This exponential increase is called amplification in the DNA Polymerase process.





Research and Development

PCR - The Underlying Technology
Functions of Components in a PCR Reaction Each component in a PCR reaction has a different function. Template DNA is essential to DNA replication. The first double stranded DNA molecule is separated, then each strand is used to create two double stranded DNA molecules that are identical to the first. Primers are used to copy specific DNA sequences, to produce two double stranded DNA molecules from the two strands of one double stranded DNA molecule. Each of two primers attach to one of the DNA strands, on the opposite side and other strand than the other. Primers are important because they allow specific DNA sequences to be targeted. Taq polymerase's function is to attach to each strand of DNA near the primer. Then the taq polymerase begins to add nucleotides. Deoxyribonucleotides are the building blocks of DNA.The polymerase attaches these nucleotides to the end of a primer to turn one strand of DNA into a double stranded DNA molecule.

What Happens During Thermal Cycling During the first step, the components are heated to 95 degrees Celsius for three minutes. Then the components are heated at 95 degrees Celsius for another thirty seconds. During this time, the two strands of DNA separate, or denature. Then the components cool to 57 degrees Celsius, when the two primers bind, or anneal. Each primer binds to a different strand of DNA, at opposite sides of the strands. Then the components are heated to 72 degrees Celsius for thirty seconds, when the DNA polymerase binds to the end of the primers, and begins extending along the DNA strand. This process is completed as the temperature is kept stable at 72 degrees Celsius for three minutes, while the polymerase attaches the remaining nucleotides to complete the two double stranded DNA molecules. During the final hold, the components are cooled to 4 degrees Celsius. During this time, the replication is complete and two double stranded DNA molecules have formed.

Which Bases Anneal to Which Bases Only certain bases can attach to eachother. There are four bases, including Adenine, Thymine, Cytosine, and Guanine. Adenine and Thymine can only bond with one another. Likewise, Cytosine and Guanine can only bond with one another.

When Base Pairing Occurs Base pairing occurs during two steps of the PCR process. It occurs during both the forth and fifth steps, when the components are heated to 72 degrees Celsius for the first thirty seconds, then for three minutes. This is because at this point, both the primer and the DNA polymerase have attached to the DNA strands, allowing for nucleotides to be attached to the DNA strands by the polymerase. During the forth stage, the polymerase attaches to the DNA strand and begins base pairing, but an additional three minutes at the same temperature of 72 degrees Celsius during stage five are necessary to finish up the process.