BME100 s2015:Group4 12pmL4

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Contents

OUR TEAM

Name: Sydney WallaceRole(s)
Name: Sydney Wallace
Role(s)
Name: nima.sadeghiRole(s)
Name: nima.sadeghi
Role(s)
Jonathan MoronesoRole(s)
Jonathan Moroneso
Role(s)
Name: Fatimah Al habibRole(s)
Name: Fatimah Al habib
Role(s)

LAB 4 WRITE-UP

Protocol

Materials

  • Lab Coat
  • Disposable Gloves
  • Micropipettor
  • Disposable Pipet Tips
  • Cup for Pipet Tips
  • PCR Master Mix
  • DNA and Primer Mix
  • Open PCR Machine
  • Ice Bucket
  • Ice


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G# + Positive control none
G# - Negative control none
G# 1-1 Patient 1, replicate 1 16819
G# 1-2 Patient 1, replicate 2 16819
G# 1-3 Patient 1, replicate 3 16819
G# 2-1 Patient 2, replicate 1 59134
G# 2-2 Patient 2, replicate 2 59134
G# 2-3 Patient 2, replicate 3 59134


DNA Sample Set-up Procedure

  1. Put on lab coat and gloves.
  2. Prepare ice bucket with ice.
  3. Thaw the PCR Master Mix tubes and DNA/Primer mix tubes at room temperature.
  4. Vortex and then briefly micro-centrifuge each tube.
  5. Prepare each PCR tube with appropriate label as found on the PCR Reaction List Table.
  6. Pipet 50μl of Master Mix and 50μl of DNA/Primer mix into each corresponding tube. (Total volume of the third tube is now 100μl.) Immediately place the finished tube into the ice before moving onto the next tube.
  7. Place the ready tubes into the thermocycler.


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






Research and Development

PCR - The Underlying Technology

Q1. What is the function of each component of a PCR reactant? Template DNA: DNA that holds the portion of DNA to be selected and copied in the PCR reaction. Primers: Primers tell the Taq Polymerase where to connect to the DNA strand and start extending, and where to stop. Select the region to be copied. Primers connect to a specfic region on DNA, since primers are designed in the lab, the research controls which region the primers bind to. Taq Polymerase: The enzyme that attaches to the primer and attaches the dNTP's to the selected region of the DNA. Deoxyribonucleotides (dNTP’s): Free floating pieces of DNA, which including the phosphate, sugar, and base that make up DNA; A, T, G, C. These are used up and connected to the template DNA to make the copies.

Q2. What happens to the components (listed above) during each step of thermal cycle? INITIAL STEP: 95°C for 3 minutes: This steps denatures the DNA for the first time. It causes each piece of DNA to split into single stranded pieces of DNA. The temperature allows the bonds to break. Denature at 95°C for 30 seconds: This is each subsequent denaturing of the DNA strands. All strands will split into two, single stranded components. The temperature allows the bonds to break. Anneal at 57°C for 30 seconds: The decrease in temperature allows the primers to anneal to each single strand of DNA, at both the starting and ending points of the pre-determined region. Extend at 72°C for 30 seconds: This temperature increase allows the optimal conditions for Taq Polymerase to attach the dNTP’s to the existing single-stranded DNA. FINAL STEP: 72°C for 3 minutes: The final hold allows all single stranded DNA to be paired, so that all the DNA is now back to double-stranded. FINAL HOLD: 4°C: The final hold keeps the DNA intact, and ready for removal and the next step in the procedure.

Q3. Which base anneals to each base listed below? A binds to T, T binds to A, C binds to G, and G binds to C.

Q4. During which two steps of thermal cycling does base pairing occur? Explain your answer. These two steps are annealing and extension. Annealing allows the primers to attach to the single stranded DNA sections, due to the given temperature. In the next step of extension, the base-pairs are paired with their counterparts, and the complete DNA strand is then formed. The temperature at this step is optimal for Taq Polymerase.


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