BME100 f2014:Group19 L4

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Contents

OUR TEAM

Name: Andrew Carlson
Name: Andrew Carlson
Name: Stacy Stoddard
Name: Stacy Stoddard
Name: Gareth Palas
Name: Gareth Palas
Name: Josh Hislop
Name: Josh Hislop
Name: Josh Martin
Name: Josh Martin
Name: Jose Elenes
Name: Jose Elenes

LAB 4 WRITE-UP

Protocol

Materials

  • Lab Report and disposable gloves
  • PCR mix, 8 tubes, 50 µL each: Mix contains Taq DNA Polymerase, MgCl ~2~, and dNTP's
  • DNA/primer Mix, 8 tubes, 50 µL each: Each mix contains different template DNA. All tubes have the same forward and reverse primer
  • A strip of empty PCR tubes
  • Disposable pipette tips
  • Cup for discarded tips
  • Micropipetter
  • OpenPCR machine


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G19 + Positive control none
G19 - Negative control none
G19 1-1 Patient 1, replicate 1 58961
G19 1-2 Patient 1, replicate 2 58961
G19 1-3 Patient 1, replicate 3 58961
G19 2-1 Patient 2, replicate 1 79602
G19 2-2 Patient 2, replicate 2 79602
G19 2-3 Patient 2, replicate 3 79602


DNA Sample Set-up Procedure

  1. Gather materials and lab equipment for experiment
  2. Open and label each of empty PCR tubes according to the sample that will be placed inside them
  3. In each of the tubes, micropipette 50 µL of the PCR mix
  4. Discard micropipette tip and replace with clean one
  5. Then micropipette 50 µL of the DNA/primer mix to each tube
  6. Discard micropipette tip and replace with clean one
  7. Finally micropipette each of the samples given into the corresponding tube
  8. Close the lids of each PCR tube
  9. Place the tubes into the PCR machine and run for 35 cycles


OpenPCR program

Thermal Cycling follows the following steps:

  1. Heat the lid to 100 ° C
  2. Heat up the sample to 95 ° C for 2 minutes
  3. Denature the sample for an additional 30 seconds to separate strands of DNA
  4. Anneal the strands of DNA at 57 ° C for another 30 seconds
  5. Extend or link the two pieces of DNA at 72 ° C for 30 seconds
  6. Repeat steps 3-6 for 35 cycles
  7. Keep the PCR at 72 ° C for another 2 minutes
  8. Set the final hold at 4 ° C

The purpose of this cycling is to heat the DNA so it's strands separate, then introduce new DNA primers to create the DNA sequence that is desired. These primers act as a beginning and ending point for the base pairing. The DNA is then multiplied several times until all that is being replicated is the desired DNA sequence.






Research and Development

PCR - The Underlying Technology

In a Polymerase Chain Reaction (PCR), multiple components need to be mixed together in order for the desired reaction to occur. The first component is template DNA, which contains the DNA target that needs to be amplified. Next are primers, short pieces of lab-made DNA that attach to the segments of DNA that need to be copied. Taq Polymerase is an enzyme that binds to the DNA strand and adds nucleotides, creating whole DNA from the single strand. Nucleotides are the individual components<of DNA. They are a sugar and they pair A-T C-G.

PCR occurs in only six steps. The initial step prepares the DNA for the polymerase in a process called hot-start PCR (other types of PCR eliminate this step).

The next step, called denaturation, heats the sample to nearly its boiling point to "melt" the DNA into single strands. The components of the reaction are heated to 95°C; the double helix structure of the template DNA unravels, and the strands are broken apart as the energy of the strands becomes too great for hydrogen bonds to hold together. It is for this same reason that the primers and deoxyribonucleotides (dNTP's) float free; the hydrogen bonds that would normally bind the primers to the strands cannot hold at such a high temperature. With no primers bonded to allow the Taq Polymerase to replicate, they float free as well.

Next, in the Annealing step, the PCR machine cools to about 50°C. During annealing, the components are cooled to allow primers to bind to the separated template DNA strands. At this point, some of the Taq Polymerase can begin to extend the replicated strands of DNA, adding on the dNTP's to create the new strand.

The temperature is adjusted during the extension step to the ideal temperature for Taq Polymerase activity. It's at this phase and temperature that the template DNA is replicated most efficiently. Once again, the dNTP's are used by the Taq Polymerase to create the complementary strand to the template DNA. An entire strand of DNA is synthesized from each half of the template strand.

The final hold occurs at 4°C. This allows the double helix to re-form and preserves the DNA for an indefinite amount of time.

DNA binds in the following manner: Thymine-Adenine, Cytosine-Guanine (TACG)

Base pairing occurs in the annealing step and the extension step. In the annealing step, the primer binds to the template using base pairing. In extension, polymerase pairs nucleotides with their correspondent in the single strand.



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