BME100 s2015:Group5 9amL4

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Contents

OUR TEAM

Name: Jacob Licko
Name: Jacob Licko
Name: Stephen Belikoff
Name: Stephen Belikoff
Name: Khalil Khoury
Name: Khalil Khoury
Name: Daniela Romero
Name: Daniela Romero
Name: Wei Lu
Name: Wei Lu

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 (http://www.promega.com/resources/protocols/product-information-sheets/g/gotaq-colorless-master-mix-m714-protocol/)
  • 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
  • Open PCR machine: shared by two groups


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G5 P + Positive control none
G5 N - Negative control none
G5 1-1 Patient 1, replicate 1 59697
G5 1-2 Patient 1, replicate 2 59797
G5 1-3 Patient 1, replicate 3 59797
G5 2-1 Patient 2, replicate 1 79049
G5 2-2 Patient 2, replicate 2 79049
G5 2-3 Patient 2, replicate 3 79049


DNA Sample Set-up Procedure

  1. Gather all required materials (listed above)
  2. Divide strip of 8 empty tubes into two strips of four linked tubes
  3. Follow the instructions from the Student Workbook and label each of the 8 tubes legibly with a black fine point marker in the following way:
  1. G (Group)
  2. # (Group Number)
  3. Patient # (1 or 2) [DASH] Sample # (1, 2, or 3)
 ((Two of the tubes must be labelled as "control." [P or N]))
  • Patient Example: G5 1-2 (Group 5, Patient 1, Sample 2)
  • Control Example: G5 P (Group 5, Positive Control)
  1. Place PCR tubes into the rack
  2. Set the volume the micropipettor will extract to 50 μL
  3. Press the discharge end of the micropipettor onto an unused tip and lift
  4. Press down plunger to the first position and insert the tip into the PCR reaction mix, just below the surface of the liquid
  5. Gradually release the plunger and extract 50 μL of the PCR reaction mix from the tube
  6. Remove tip from solution, wait two seconds, and then discharge all 50 μL of the PCR reaction mix into the tube (gradually push plunger down to second position) in the rack that corresponds with the patient and sample number that is being prepared for a Polymerase Chain Reaction (i.e. discharge into tube G5 1-2 for patient 1 sample 2)
  7. Remove tip from tube, wait two seconds, and then discard the used micropipettor tip into a biological waste container (cup)
  8. Press the discharge end of the micropipettor onto an unused tip and lift
  9. Press down the plunger to the first position and insert the tip into the DNA primer mix, just below the surface of the liquid
  10. Gradually release the plunger and extract 50 μL of the DNA primer mix from the tube
  11. Remove tip from solution, wait two seconds, and then discharge all 50 μL of the DNA primer mix into the tube that is currently being prepared
  12. Remove tip from tube, wait two seconds, then discard the used micropipettor tip into a biological waste container (cup)
  13. Set aside the micropipettor and close the lid of the tube containing the prepared sample tightly. It should click audibly when sealed completely
  14. Repeat Steps 5-16 for each of the remaining tubes
  15. Once all of the tubes have all been prepared, insert the two strips of four linked tubes into the PCR machine
  16. Allow the TA to operate the PCR machine


OpenPCR program

  1. HEATED LID: 100°C
  2. INITIAL STEP: 95°C for 2 minutes
  3. NUMBER OF CYCLES: 35
  1. Denature at 95°C for 30 seconds
  2. Anneal at 57°C for 30 seconds
  3. Extend at 72°C for 30 seconds
  1. FINAL STEP: 72°C for 2 minutes
  2. 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: This is the sequence of DNA that is intended for amplification. It will undergo extreme heat in order for it to denature.

Primers: A primer is a single strand of DNA that serves as the starting point for DNA synthesis that binds to specific DNA sequences.

Taq Polymerase: This is a thermostable enzyme that recombines nucleotides following separation by heat. It creates a new, complimentary strand of DNA through adding new nucleotides to the old ones.

Deoxyribonucleotides (dNTP's): There are the building blocks of DNA: adenine (A), thymine (T), cytosine (C) and guanine (G). These are used by the Taq Polymerase to synthesize the complementary strand of DNA.

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

Initial step (95°C for 2 minutes): The temperature of the reaction is increased to 95°C and held for two minutes in preparation for separation.

Denature at 95°C for 30 seconds: The double stranded DNA in the reaction is broken down into single strands of DNA for amplification.

Anneal at 57°C for 30 seconds: The temperature of the reaction is lowered to around 5°C below the melting point of the primer (57°C) so that the primers will bind to specific points on the template DNA that indicate areas that will be amplified.

Extend at 72°C for 30 seconds: The temperature of the reaction is increased to the ideal temperature for polymerase activity (72°C) so that the Taq polymerase will bind to the primers that are already on the template DNA and will begin to synthesize the complimentary strand.

Final step (72°C for 3 minutes): These three minutes are used to make sure that each strand has been fully extended.

Final hold (4°C): This is the temperature to deactivate the Taq polymerase and end the process.

Q3: DNA is made up of four types of molecules called nucleotides, designated as A, T, C and G. Base-pairing, driven by hydrogen bonding, allows base pairs to stick together. Which base anneals to each base listed below?;;;

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

Q4: During which two steps of thermal cycling does base-pairing occur? Explain your answers.

Base-pairing occurs during the extended and "final step" parts of PCR. Taq polymerase is optimized during the extended phase and can attach to the primers of the template DNA strands, and during the final step the proper base pairs are gathered and attached to their correct partners.






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