BME100 s2015:Group1 9amL4

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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: Grace Kim
Name: Jonathan Moreno
Name: Shawn Striker
Name: student
Role(s)
Name: student
Role(s)
Name: student
Role(s)

LAB 4 WRITE-UP

Protocol

Materials

  • Lab coat
  • Disposable gloves
  • PCR reaction mix
  • DNA/primer mix
  • 16 tubes (50 micro-liters)
  • Strip of empty PCR tubes
  • Disposable pipette tips
  • Cup for discarded pipette tips
  • Micropipettor
  • OpenPCR machine


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 11316
G1 1-2 Patient 1, replicate 2 11316
G1 1-3 Patient 1, replicate 3 11316
G1 2-1 Patient 2, replicate 1 25353
G1 2-2 Patient 2, replicate 2 25353
G1 2-3 Patient 2, replicate 3 25353


DNA Sample Set-up Procedure

  1. Gather materials
  2. Set aside patients' DNA samples and PCR reaction mix
  3. Cut the strip of empty PCR tubes to create two strips of four linked tubes
  4. Label the sides of the empty tubes appropriately
  5. Place the PCR tubes in a rack
  6. Place a new pipette tip onto the micropipettor
  7. Extract 50 μL of PCR reaction mix
  8. Release PCR reaction mix into corresponding PCR tube
  9. Discard pipette tip into cup
  10. Place a new pipette tip onto the micropipettot
  11. Extract 50 μL of DNA/primer mix
  12. Release DNA/primer mix into the PCR tube used in Step 8
  13. Repeat Steps 6-12 for the rest of the tubes
  14. Close lids tightly
  15. Place the tubes in the PCR machine


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
-Extend at 72°C for 30 seconds

  • Final Step: 72°C for 2 minutes
  • Final Hold: 4°C




Research and Development

PCR - The Underlying Technology

Q1. What is the function of each component of a PCR reaction?

  • Template DNA: desired segment of the DNA intended for amplification
  • Primars: attach to either end of the DNA segment that is being replicated
  • Taq Polymerase: reads DNA code and than attaches matching nucleotides to complete the DNA replication
  • dNTP's: building blocks of DNA

Q2. What happens to the components (listed above) during each step of thermal cycling?

  • Initial Step (95 degrees celsius): the DNA strand is Heated to 95 degrees celsius
  • Denature (95 degrees celsius): DNA separated into two single DNA strands
  • Anneal (57 degrees celsius): Primer connects to designated targets before DNA strands pair back together
  • Extend (72 degree celsius): triggers the DNA polymerase which locates and connects to the primer on the DNA strand at either end
  • Final Step (72 degrees celsius): complementary nucleotides are added to the strand
  • Final Hold (4 degrees celsius): Taq polymerase is deactivated. 4 separate strands of DNA are left and the reaction takes place again to make two complete DNA strands, and so forth

Q3. Which base anneals to each base listed below?

  • Adenine (A): T
  • Thymine (T): A
  • Cytosine (C): G
  • Guanine (G): C

Q4. During which two steps of thermal cycling does base-pairing occur?

  • Extended and Final Step. Taq polymerase is at the optimal temperature of 72 degrees celsius and can attach to primers on template DNA strands. Corresponding base pairs are then gathered to create a complementary DNA strand for the amplification process.