BME100 f2014:Group10 L4

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OUR TEAM

Name: Meredith Bothman
Name: Dawei Jiang
Name: Cera Lange
Name: Steven Nguyen
Name: Eun Duk Seo

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
  • Cup for discarded tips
  • Micropipettor
  • OpenPCR machine: shared by two groups


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G10 + Positive control none
G10 - Negative control none
G10 1-1 Patient 1, replicate 1 62548
G10 1-2 Patient 1, replicate 2 62548
G10 1-3 Patient 1, replicate 3 62548
G10 2-1 Patient 2, replicate 1 14376
G10 2-2 Patient 2, replicate 2 14376
G10 2-3 Patient 2, replicate 3 14376


DNA Sample Set-up Procedure

  1. Gather all materials needed for the lab.
  2. Take the strips of empty PCR tubes that were provided, and cut the strip in half to ensure that the tubes will fit in the thermal cycler. The tubes were labeled as follows:
Tube 1 G10+
Tube 2 G10-
Tube 3 G10 1-1
Tube 4 G10 1-2
Tube 5 G10 1-3
Tube 6 G10 2-1
Tube 7 G10 2-2
Tube 8 G10 2-3
  1. Label the sides of the PCR tubes with tube labels corresponding to the patient number, trial number and group number.
  2. Place the PCR tubes in a holding rack
  3. Beginning with the empty PCR tube labeled as the positive control, transfer 50 μL of PCR reaction mix to the tube using proper pipetting technique. Discard the tip to avoid cross-contamination between tubes.
  4. Transfer the positive control DNA/primer mix to the same PCR tube to make the total volume 100 μL.
  5. Repeat steps 5 and 6 for all remaining tubes. Make sure that all tubes contain both DNA/primer mix and PCR mix for a total of 100 μL.
  6. Make sure that the lids are sealed on the PCR tubes.
  7. Place the tubes in the assigned open PCR machine. Start the machine once all 16 slots are filled.


OpenPCR program

The thermal cycling program should be set to specific settings before the commencement of the OpenPCR reaction.

  • Lid heated to 100°C
  • Initial Step- 2 minutes at 95°C
  • 35 cycles of:
    • Denature- 30 seconds at 95°C
    • Anneal- 30 seconds at 57°C
    • Extend- 30 seconds at 72°C
  • Final Step- 2 minutes at 72°C
  • Final hold at 4 °C






Research and Development

PCR - The Underlying Technology

Ingredients to a PCR Reaction

The success of a PCR reaction depends on a specific mix of ingredients. The first is the DNA to be amplified, which serves as the template for the rest of the reaction. The second is the primers used within the reaction. The primers keep the two strands of DNA from reattaching during the annealing stage and also provide a bonding site for the DNA polymerase to begin synthesis. After several cycles, the primers also serve as the endpieces to the newly synthesized DNA fragments. Taq polymerase is also included within the mix of components. It is the bonding agent which pairs the dNTP's with the nucleotides of the template DNA. The last piece of the mixture is deoxyribonucleotides, or dNTP's. dNTP's are the free bases which are paired with the bases on the template DNA strand to produce the second half of a synthesized DNA strand.

The Process of Thermal Cycling

During the process of thermal cycling, the ingredients undergo a series of temperature changes to facilitate the production of more DNA. The mixture is initially heated to a temperature of 95 °C for a period of 3 minutes. This near boiling temperature causes the double helix of the DNA to separate to create two single-stranded DNA molecules. This initial step prepares the DNA for a series of 35 repetitions of the same three steps. These three steps include denaturing, annealing, and extension. During the denaturing step, the mixture is heated to 95 °C for 30 seconds. This has the same effect as the initial step in that it causes the double-stranded DNA to separate. The mixture is then cooled to 57 °C for 30 seconds so that annealing, or binding, can take place. The single-stranded DNA attempts to reconnect with its partner strand in the cooler temperature, but it is outnumbered by the primers that were added to the mixture. The primer sequences then bond to the DNA strands to keep them from rejoining and to provide markers for the next step. The mixture is then heated to a temperature of 72 °C for the extension step. During the extension step, the taq polymerase bonds to the primer sequences and pairs the dNTP's to the base pairs of the template DNA sequence. This produces two double-stranded DNA sequences from the initial template. After the 35 steps are completed, the mixture is held at 72 °C for 3 minutes to complete the extension process. The final product is then held at a temperature of 4 °C until it is removed from the thermal cycler and either refrigerated or subjected to further tests.

Base-Pairing

The process of base-pairing is necessary to the PCR reaction. There are four types of DNA nucleotides: adenine, thymine, guanine, and cytosine. These molecules are paired through the use of hydrogen bonding. The dNTP's are paired with the existing strand of template DNA according to the following formula: adenine pairs with thymine and guanine pairs with cytosine. Base-pairing occurs during both the annealing and extending steps of thermal cycling. During the annealing step the specific pattern of nucleotides found within the primer sequences bond to their complementary sequences within the template DNA. This is made possible due to the strict rules of base-pairing. Base-pairing also occurs during the extension stage, when taq polymerase is used to pair dNTP's to their complementary bases on the template DNA sequence.