BME100 f2014:Group22 L4

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Contents

OUR TEAM

Name: Justin Else
Name: Justin Else
Name: Aaron Raber
Name: Aaron Raber
Name: Chase Radigan
Name: Chase Radigan
Name: Daniel Sorto
Name: Daniel Sorto
Name: Zach Steidl
Name: Zach Steidl
Name: Lisa Lavergne
Name: Lisa Lavergne

LAB 4 WRITE-UP

Protocol

Materials

  • Lab coat and 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 ti
  • Cup for discarded tips
  • Micropipettor
  • OpenPCR machine: shared by two groups


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G22 + Positive control none
G22 - Negative control none
G22 1-1 Patient 1, replicate 1 86342
G22 1-2 Patient 1, replicate 2 86342
G22 1-3 Patient 1, replicate 3 86342
G22 2-1 Patient 2, replicate 1 34714
G22 2-2 Patient 2, replicate 2 34714
G22 2-3 Patient 2, replicate 3 34714


DNA Sample Set-up Procedure

  1. Prepare samples of extracted DNA by pipetting (with a proper micropipettor tip) a sample of the extracted DNA from the chilled vial into a PCR tube, discarding the tip after use.
  2. Add Primer 1 to the DNA solution already in the PCR tube by pipetting the Primer 1 solution into the PRC tube, using a disposable tip as with the DNA solution.
  3. Add Primer 2 to the PCR tube in the same fashion as Primer 1.
  4. Add a solution of free nucleotides to the PCR tube in the same fashion as described above with Primers 1 and 2.
  5. Add a solution of polymerase enzyme solution into the PCR tube.
  6. Close the cap to the PCR tube and place it in a PRC tube slot in the thermocycler , closing the lid and starting the thermocycler and running it for [X] cycles.

From PCR Virtual Lab, a simulation on the Learn.Genetics site at the University of Utah website.

OpenPCR program

Screen capture of "Polymerase Chain Reaction (PCR)" video from http://openpcr.org/what-is-pcr





Research and Development

PCR - The Underlying Technology

What Are the Components of a PCR Reaction?

PCR consists of a solution of sample DNA to be amplified, as well as primer molecules, Taq Polymerase enzymes, and deoxyribonucleotides, or dNTP's. The template DNA can be any DNA extracted from a patient or organism to be tested; this DNA will be processed, and specific sequences of DNA will be isolated and amplified many times so that it can be detected by fluorescent markers if it exists in the sample. In a PCR reaction, the primers mark the beginning of DNA replication and, if the target sequence exists in the sample DNA, the primers bind to a specific sequence of nucleotides that marks the beginning of the target sequence. The primers mark the entry point for Taq Polymerase, an enzyme that begins at a primer and reads from the 3' end of the DNA to the 5' end, assembling a new strand of DNA from free deoxyribonucleotides (dNTP's), the monomers (building blocks) of DNA.

From PCR Virtual Lab, a simulation on the Learn.Genetics site at the University of Utah website

What Happens During Each Step of Thermal Cycling?

During a PCR thermal cycle, the mixture of DNA, dNTP's, and primers are first heated to around 95°C. During this time, the DNA molecules are denatured, splitting into single strands. Next, the cycle cools the PCR tubes and their mixtures down to 57°C, allowing the DNA primers to attach to their respective places on the DNA (annealment), marking it for replication by DNA Taq Polymerase. Next, the mixture is heated slightly to 72°C, which allows the polymerase to bind to the primer sites and begin copying the DNA (extension).

Which Nucleotides Bond to Each Other?

<p> Adenosine (A) binds to Thymine (T)

Cytosine (C) binds to Guanine (G)




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