BME100 f2016:Group4 W1030AM L4

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Contents

OUR TEAM

Matt Morton
Matt Morton
Mahina Wing
Mahina Wing
Trevor Wood
Trevor Wood
Jaeger Moore
Jaeger Moore

LAB 4 WRITE-UP

Protocol

Materials

  • Lab coat and disposable gloves
  • PCR reaction mix, 8 tubes, 50 μL each: Mix contains Taq DNA polymerase, MgCl2, and dNTP's
  • DNA/ primer mix, 8 tubes, 50 μL each: Each mix contains a different template DNA. All tubes have the same forward primer and reverse primer.
  • A strip of empty primer tubes
  • Disposable pipette tips: only use each only once. Never reuse disposable pipette tips. If you do, the samples will become cross-contaminated
  • Cup for discarded tips
  • Micropipettor
  • OpenPCR machine: shared by two


PCR Reaction Sample List

Tube Label PCR Reaction sample Patient ID
G4 P Positive control none
G4 N Negative control none
G4 1-1 Patient 1, Replicate 1 54887
G4 1-2 Patient 1, Replicate 2 54887
G4 1-3 Patient 1, Replicate 3 54887
G4 2-1 Patient 2, Replicate 1 26612
G4 2-2 Patient 2, Replicate 2 26612
G4 2-3 Patient 2, Replicate 3 26612


DNA Sample Set-up Procedure

  1. Move extracted DNA from Patient 1 into special PCR tube via pipettor
  2. Add Primer 1 to PCR tube
  3. Add Primer 2 to PCR tube
  4. Add nucleotides to PCR tube
  5. Add DNA Polymerase to PCR tube
  6. Repeat steps 1-5 for extracted DNA from Patient 2
  7. Place PCR tubes into thermal cycler


OpenPCR program Heated Lid: 100°C

Initial Step: 95°C for 2 minutes

Number of Cycles: 25 Denature at 95°C for 30 seconds, Anneal at 57°C for 30 seconds, and Extend at 72°C for 2 minutes

Final Hold: 4°C






Research and Development

PCR - The Underlying Technology


---What is the function of each component of a PCR Reaction?---

The 4 components of a PCR reaction are Template DNA, Primers, Taq Polymerase, and Deoxyribonucleotides (dNTP's). The template DNA will be tested for a specific sequence. The Primers will attach to the beginning and end of the desired DNA sequence form the DNA template. Taq Polymerase will replicate DNA by adding complimentary nucleotides, called dNTP's, to the strand.


---What happens to the components during each step of thermal cycling?---

During the initial step of thermal cycling, the DNA strands separate and uncurl from their double helix structure. This separation continues into the Denature step of thermal cycling. Next, during the Anneal step, the temperature drops low enough where the primers are activated. They bind to the targeted sequences of DNA. The next step is the Extend step. Here, Taq Polymerase begins to add complimentary dNTP's starting at the primers and continues until the strand ends. During the Final Step, all the DNA is extended and then stored at a very low temperature in the Final Hold.


---Which base anneals to each base listed below?---

Adenine(A) - Thymine

Thymine(T) - Adenine

Cytosine(C) - Guanine

Guanine(G) - Cytosine


---During which two steps of thermal cycling does base-pairing occur?---

The first step of base-pairing in thermal cycling occurs during the Anneal stage. The primer base pairs to a specific site of the targeted DNA sequence. The second is the Extend step. Here, base pairs are added individually by Taq Polymerase from the Primer to the end of the strand.

---Illustration---

PCR illustration



SNP Information & Primer Design

Background: About the Disease SNP

The SNP disease first and foremost is a genetic disease that is cause by a single point mutation. The SNP diseases' point mutation lies in an area of the genome that codes for the protein Ankyrin 2 a member of the ankyrin family of proteins. These proteins are critical in a few cell functions such as cell motility, activation, proliferation, contact and the maintenance of specialized membrane domains. With this SNP point mutation occurring all of those functions would be hindered and not occur. In addition,SNP disease point mutations of this gene causes long QT syndrome 4 and cardiac arrhythmia in patients.

Primer Design and Testing

When the non-diseased forward and reverse primers were included in the experiment, a 220 base pair sequence result was amplified. In this case, the experiment was a success. This shows that non diseased DNA was included in the DNA sample. When the diseased forward primer was included in the experiment, no viable sections of DNA was amplified. This result suggests that the mutated ANK2 gene was not present in the DNA sample.

Non-Diseased Forward Primer
Non-Diseased Forward Primer

Diseased Forward Prime
Diseased Forward Primer

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