BME100 f2014:Group28 L4

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Owwnotebook icon.png BME 100 Fall 2014 Home
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OUR TEAM

Name: Andrew W. Hamidy
Name: Mohammed Alhusayni
Name: Brandt Hansen
Name: Kyle Brague
Name: Diego E. Reyes
Name: Kyle Henriksen

LAB 4 WRITE-UP

Protocol

Materials

samples will be cross-contaminated

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


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G28 + Positive control 76447
G28 - Negative control 58040
G28 1-1 Patient 1, replicate 1
G28 1-2 Patient 1, replicate 2
G28 1-3 Patient 1, replicate 3
G28 2-1 Patient 2, replicate 1
G28 2-2 Patient 2, replicate 2
G28 2-3 Patient 2, replicate 3


DNA Sample Set-up Procedure

  1. Get the patient samples as well as the positive and negative controls from TA. Do not open the tubes just yet. Each set of samples has a unique number associated with it known as the patient ID number. There should be three replicates of each sample.
  2. Create labels that will be used to distinguish tubes. For example G51P, or G51N, G51 1-1. Etc.
  3. Make sure to record a key to each tube for future reference.


OpenPCR program

    HEATED LID:100°C
    INITIAL STEP: 95°C for 2 minutes
    NUMBER OF CYCLES: 35
       Denature at 95°C for 30 seconds, Anneal at 57°C for 30 seconds,and Extend at 72°C for 30 sec
    FINAL STEP: 72°C for 2 minutes
    FINAL HOLD: 4°C






Research and Development

PCR - The Underlying Technology
Function of Components

  • Primers- Pieces of DNA constructed in a lab to specifically locate a certain sequence of DNA in which is to be copied. These primers are usually 20 nucleotides long to locate the desired sequence in DNA.
  • Primase- In nature, this is what starts off PCR instead of lab created primers.
  • DNA Polymerase- Is a naturally occurring complex of proteins that attaches to the DNA and copies it with nucleotides before it is split into two. When used with a primer, it attaches itself to the end of the primer and starts to add nucleotides to complete the double strand of DNA.
  • Nucleotides- Building blocks of DNA that include Adenine, Cytosine, Guanine, and Thymine (A,C,G,T). These nucleotides are grabbed by the DNA Polymerase and attached to the primer which completes the DNA strand.

This information was taken from http://learn.genetics.utah.edu/content/labs/pcr/*



What happens to the components (listed above) during each step of thermal cycling?
INITIAL STEP: 95°C for 3 minutes: The DNA starts to lose it's double helix structure, becoming closer to a ladder.
Denature at 95°C for 30 seconds: The DNA separates into two strands.
Anneal at 57°C for 30 seconds: Primers bind to complementary matches on the target sequence
Extend at 72°C for 30 seconds: Taq polymerase binds to the primed sequences and adds nucleotides to the strand.
FINALSTEP: 72°C for 3 minutes: New sets of DNA are made swiftly without destruction of the double helix
FINAL HOLD: 4°C: Allows the machine to shut off completely and complete the reaction

This information was taken from http://openpcr.org/what-is-pcr

Adenine anneals to thymine. Thymine anneals to adenine. Guanine anneals to cytosine. Cytosine anneals to guanine.

This information was taken from http://learn.genetics.utah.edu/content/labs/pcr/*