BME100 s2015:Group10 12pmL4

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Contents

OUR TEAM

Name: Joseline Valenzuela  Experiment  Procedure
Name: Joseline Valenzuela
Experiment Procedure
Name: Itai Kreisler Data Analysis
Name: Itai Kreisler
Data Analysis
Name: Clayton NunnRole(s)
Name: Clayton Nunn
Role(s)
Name: Isaac Clouse  Data Analysis)
Name: Isaac Clouse
Data Analysis)

LAB 4 WRITE-UP

Protocol

Materials

  • Lab coat
  • Disposable gloves
  • PCR reaction mix, 8 tubes, 50 μL each
  • DNA/ primer mix, 8 tubes, 50 μL each
  • A strip of empty PCR tubes
  • Disposable pipette tips
  • Cup for discarded tips
  • micropipettor
  • OpenPCR machine


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 67713
G10 1-2 Patient 1, replicate 2 67713
G10 1-3 Patient 1, replicate 3 67713
G10 2-1 Patient 2, replicate 1 89702
G10 2-2 Patient 2, replicate 2 89702
G10 2-3 Patient 2, replicate 3 89702


DNA Sample Set-up Procedure

  1. Step 1: Obtain the necessary equipment from the supervisor.
  2. Step 2: Label all your tubes indicating the patient and the trial number, and a positive and negative control.
  3. Step 3: Pipet the patient samples into the appropriate corresponding test tube. Make in each tube there is the DNA/Primer mix, and patient samples/control. The total volume should be 100ul in each tube. Be sure to not cross contaminate the samples with used tips.
  4. Step 4: Once all your samples have the right mixes in each of them, place the tubes in the thermocycler.

OpenPCR program


Image:PCR steps group 10 PM.jpg

Image:TEAM_10_PM_PCR.jpg



Inspired by: http://users.ugent.be/~avierstr/principles/pcr.html





Research and Development

PCR - The Underlying Technology

Denaturation: Before the DNA strands are put into the Heated lid, the template strand, the strand which has the portion to be copied, is preheated to 100C. The DNA strands are then heated to a high temperature of about 95C. After thirty seconds the hydrogen bonds weaken which hold the double helix together, at which point the bonds become so weak, the strands physically separate. The dNTP's, or deoxyribonucleotide base pairs, will be exposed and the DNA is ready for primers to be attached.

Annealing: At a lower temperature, of about 50C and the time has been another thirty seconds, the primers, the starting points for copying that assist enzymes, are introduced into the mix. Since the DNA base pairs are exposed, the primers will be able to bind to specifically selected sites on the DNA. After this portion, the replication will begin.

Extension: Taq polymerase, a special kind of replicating enzyme for DNA, begins to line up the new base pairs at a temperature of 72C and after another thirty seconds has passed. At this period of time, the DNA will have been doubled. The previous three steps will be repeated 35 times in order to amplify a single section of the DNA to be copied millions and millions of times.

Final Elongation: The DNA is put through one last extension to ensure all the strands have matching base pairs to form complete double helices. This is done by heating the DNA to 72C and this is done for 2 minutes.

Final Hold: The DNA is stored at 4C to prevent it from denaturing again.


Credit: http://en.wikipedia.org/wiki/Polymerase_chain_reaction#Procedure




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