BME100 f2014:Group33 L4

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Contents

OUR TEAM

Name: Emigdio Ruiz Esquivel
Name: Emigdio Ruiz Esquivel
Name: John Cunningham
Name: John Cunningham
Name: Wandasun Sihanath
Name: Wandasun Sihanath
Name: Chris Adams
Name: Chris Adams
Name: Tim Snelling
Name: Tim Snelling
Name: Breanna Falcon
Name: Breanna Falcon

LAB 1 WRITE-UP

Section One: Protocol Planning

Materials

  • Lab coat and disposable gloves
  • PCR reaction mix, 8 tubes, 50uL each: Mix contains Taq DNA polymerase,MgCl2, and dNTP's

[1]

  • DNA/primer mix, 8 tubes, 50uL each: Each mix contains a different template DNA. All tubes have the same forward primer and the reverse primer
  • A strip of empty PCR tubes
  • Disposable pipette tips: only use each once. Never re-use disposable pipette tips or samples will be cross-contaminated
  • Cup for discarded tips
  • Micropipettor
  • Open PCR machine:shared by two groups


Thermal Cycler Program


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G33 + Positive control none
G33 - Negative control none
G33 1-1 Patient 1, replicate 1 31366
G33 1-2 Patient 1, replicate 2 31366
G33 1-3 Patient 1, replicate 3 31366
G33 2-1 Patient 2, replicate 1 26606
G33 2-2 Patient 2, replicate 2 26606
G33 2-3 Patient 2, replicate 3 26606

DNA Sample Set-up Procedure

  • 1. Set the micro pipette to 50 micro-liter
  • 2. Attach a fresh micro pipette tube to the micro pipette pump.
  • 3. Draw Patient DNA of 50 micro-liters
  • 4. Deposit Patient DNA into empty PCR container
  • 5. Dispose of micro pipette tip
  • 6. Attach a fresh micro pipette tube to the micro pipette pump.
  • 7. Draw PCR of 50 micro-liters
  • 8. Deposit PCR of 50 micro-liters into empty PCR container
  • 9. Dispose of micro pipette tip
  • 10. Placing the tubes in the Thermal Cycler
  • 11. Repeat steps 1-10



PCR Reaction Mix

  • Component 1
  • Component 2
  • Component 3...


DNA/ primer Mix

  • Component 1
  • Component 2
  • Component 3...


OpenPCR program The following values will be used to run a heating and cooling program on the thermal cycler.

  1. Heated Lid: 100°C
  2. Initial Step: 95°C for 2 minutes
  3. Number of Cycles: 35
  4. Denature at 95°C for 30 seconds, Anneal at 57°C for 30 seconds, and Extend at 72°C for 30 seconds.
  5. Final Step: 72°C for 2 minutes
  6. Final Hold: 4°C




Section Two: Research and Development

PCR - The Underlying Technology

Functions of a PCR Reaction

  • Template DNA: a sample of DNA that contains the targeted sequence
  • Primers: short pieces of single-stranded DNA that are complementary to a targeted sequence, allows polymerase to begin synthesizing new DNA
  • Taq Polymerase: a heat stable enzyme used in PCR to amplify DNA segments
  • Deoxyribonucleotides (dNTP’s): single units of nucleotides (A, G, T, and C), building blocks for new DNA strands


The Process of Thermal Cycling

  • Initial Step: 95 ℃ for 3 minutes, heated to near boiling
  • Denature: 95 ℃ for 30 seconds, the DNA double helix separates to create two separate strands of complementary DNA
  • Anneal: 57 ℃ for 30 seconds, primer sequences bond to DNA to keep them from rejoining
  • Extend: 72 ℃ for 30 seconds, the DNA polymerase enzyme is activate and complementary nucleotides are added to the separated DNA strands
  • Final Step: 72 ℃ for 3 minutes, bonding is secured
  • Final Hold: 4 ℃, DNA is kept at a cool temperature


Base-Pairing

Image:Base.jpg
DNA is made up of four different nucleotides: adenine, guanine, thymine, and cytosine. These four components are the building blocks of DNA and RNA. Adenine is paired with thymine by double hydrogen bonds, and guanine and cytosine are paired by a triple hydrogen bond.
Base-pairing occurs during annealing and extending in thermal cycling. During annealing, the newly denatured DNA wants to go back to it's original shape of a double helix.Only the most stable strands can do this since they are the desired ones and then they get paired off there. During extending, the DNA polymerase connects at the primers and bonds in the 5' to 3' direction, which is by definition the bonding of the base pairs.

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