BME100 s2015:Group11 12pmL4

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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: Miranda Kaml
Role(s)
Name: Framarz Alam
Role(s)
Name: Joe Florio
Role(s)
Name: student
Role(s)
Name: student
Role(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: use each only 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

PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G#11PC + Positive control none
G#11NC - Negative control none
G#11 1-1 Patient 1, replicate 1 37595
G#11 1-2 Patient 1, replicate 2 37595
G#11 1-3 Patient 1, replicate 3 37595
G#11 2-1 Patient 2, replicate 1 32179
G#11 2-2 Patient 2, replicate 2 32179
G#11 2-3 Patient 2, replicate 3 32179


DNA Sample Set-up Procedure

  1. Step 1 : Add DNA samples to separate PCR reaction tubes using different pipette tips for each sample
  2. Step 2 : Add 50μL of the DNA primer mix to each PCR tube containing a DNA sample
  3. Step 3 : Add 50μL of the PCR reaction mix to each PCR tube containing a DNA sample
  4. Step 4 : Put each of the samples into the 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°






Research and Development

PCR - The Underlying Technology

Functions of PCR Components

Template DNA This is the DNA from which the the PCR will replicate a particular sequence of interest
Primers These nucleic acids attach themselves to opposite ends of the sequence of interest once the DNA double helix has been unwound. They provide a marker to indicate where replication should begin.
Taq Polymerase This enzyme is responsible for attaching free nucleotides in a complementary order to the sequence of interest in the single DNA strand.


Thermal Cycling of PCR Components

Initial Step: 95°C for 3 minutes Solution is heated to begin denaturing of DNA molecules.
Denature at 95°C for 30 seconds Double helix of DNA molecules is unwound, leaving single strands of DNA in the solution.
Anneal at 57°C for 30 seconds Primers attach themselves to the ends of the sequence of interest in each DNA strand.
Extend at 72°C for 30 seconds Taq Polymerase attaches at the site of the DNA where the primer is.
Final Step: 72°C for 3 minutes Taq Polymerase molecules begin replication by attaching free nucleotides in a complementary order to the single-strand DNA molecules.
Final hold: 4°C Solution is cooled to allow the single-strand DNA molecules to reform into their original double helix shape, leaving many fully-formed copies of the desired sequence.