BME100 f2017:Group4 W1030 L4

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OUR TEAM

Name: Alexis Chavez
Human
Name: Jacob Schuler
Slightly Above average student
Name: Nazmina Ansari
To Be Determined
Name: Quentin Ellist
The Man the Myth the Legend
Name: Ryan Daughtry
Famous Singer

LAB 4 WRITE-UP

Protocol

Materials

  • Lab Coat and disposable gloves
  • PCR Reaction mix, 8 tubes, 50 microliters: Mix contains Taq DNA polymerase, MgCl2, and dNTP's
  • DNA/Primer mix, 8 tubes, 50 microliters 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


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G4 + Positive control G4 P
G4 - Negative control G4 N
G4 1-1 Patient 1, replicate 1 G4 1-1 (40178)
G4 1-2 Patient 1, replicate 2 G4 1-2 (40178)
G4 1-3 Patient 1, replicate 3 G4 1-3 (40178)
G4 2-1 Patient 2, replicate 1 G4 2-1 (92225)
G4 2-2 Patient 2, replicate 2 G4 2-2 (92225)
G4 2-3 Patient 2, replicate 3 G4 2-3 (92225)


DNA Sample Set-up Procedure

  1. Place the PCR reaction mix, consisting of Taq DNA Polymerase, Magnesium Chloride, and dNTP's, into the PCR tubes> Be sure to use disposable pipette tips, and replace them after ever use.
  2. Next, place the DNA/ primer mix into the PCR tubes, as these contain the different template DNA. Follow the same procedures regarding pipettes.
  3. Upon placing all of these together in the PCR tubes, place the tubes into the thermal cycler.


OpenPCR program

HEATED LID: 100 Degrees Celsius

INITIAL STEP: 95 Degrees Celsius for 2 Minutes

NUMBER OF CYCLES: 25

     Denature at 95 Degrees Celsius for 30 seconds
     Anneal at 57 Degrees Celsius for 30 seconds
     Extend at 72 Degrees Celsius for 30 seconds

FINAL STEP: 72 Degrees Celsius for 2 minutes

FINAL HOLD: 4 Degrees Celsius


Heatcycler volare ques.png






Research and Development

PCR - The Underlying Technology

Template DNA

Template DNA is the selected sequence of DNA typically between 75-1000 base pairs. It is the sequence being amplified by PCR

Primers

Primers are proteins that bind to very specific sequences within a gene. The primer then allow for a Polymerase (in the case Taq Polymerase) to bind to the primer and begin to copy the selected DNA sequence

Taq Polymerase

Taq Polymerase is a protein in the Polymerase family that allows for replication of a template strand of DNA. The reason Taq Polymerase is used instead of the body's natural DNA Polymerase is because Taq Polymerase can withstand the high temperatures required by PCR without denaturing. Taq Polymerase comes from the Thermus aquaticus bacteria.

Deoxyribonucleotides

Deoxyribonucleotides are special forms of Adenine, Guanine, Thymine, and Cytosine. The difference between these and standard nucleotides is that they are in their triphosphate form. This means that as Taq Polymerase adds these bases to the template strand the triphosphate breaks and provides energy for the polymerizing reaction. They are used to match up against the template strand of DNA and produce a complimentary strand.



SNP Information & Primer Design

Background: About the Disease SNP
The disease SNP stands for Single nucleotide polymorphism. This genetic disease occurs when a specific variation in a single nucleotide of adenine, thymine, cytosine, or guanine. These nucleotides can be changed, removed, or added to a polynucleotide sequence. These changes can have a variety of effects upon the DNA of the organism, as it can cause drastic changes in the amino acid sequences. These can affect many things, like pathogenic responses, reactions to chemicals, medications, vaccinations, and the like.

Primer Design and Testing
The primers shown below were obtained by copying 20 nucleotides ending in the mutated nucleotide for the disease primer, and the normal nucleotide for the normal primer. The reverse primer was the same for both of these pairs.

Disease Pair.jpg Diseased Pair Normal Pair.jpg Normal Pair