BME100 s2018:Group2 W0800 L4

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Owwnotebook icon.png BME 100 Spring 2018 Home
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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
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OUR TEAM

Name: Nicholas Kindseth
Role(s)
Name: Kameron Moore
Role(s)
Name: Andrew Opstad
Role(s)

LAB 4 WRITE-UP

Protocol

Materials

  • Lab Coat and disposable gloves
  • PCR reac on 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 PCR tubes
  • Disposable pipe e ps: only use each only once. Never reuse disposable pipe e ps. If you do, the samples will become cross‐contaminated
  • Cup for discarded tips
  • Micropipettor
  • OpenPCR machine: shared by two groups


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G2 + Positive control none
G2 - Negative control none
G2 1-1 Patient 1, replicate 1 37987
G2 1-2 Patient 1, replicate 2 37987
G2 1-3 Patient 1, replicate 3 37987
G2 2-1 Patient 2, replicate 1 29674
G2 2-2 Patient 2, replicate 2 29674
G2 2-3 Patient 2, replicate 3 29674


DNA Sample Set-up Procedure

  1. Extract DNA sample from hair, saliva, blood, or other source
  2. Move DNA into PCR tube using micropipette
  3. Add Primer 1 to PCR tube using micropipette
  4. Add Primer 2 to PCR tube using micropipette
  5. Add nucleotides to PCR tube using micropipette
  6. Add DNA Polymerase to the PCR tube
  7. Move PCR tube to DNA Thermocycler to heat sample


OpenPCR program

  1. Begin Cycle 1
  2. Start DNA Thermocycler, heating the sample first to 95°C
  3. Reduce temperature to 50°C
  4. Heat sample to 72°C
  5. Run Cycle 2 following steps 1-4
  6. Run Cycle 3 following steps 1-4; during this cycle the target fragments will begin to populate
  7. Run Cycle 4 following steps 1-4; during this cycle 8 target fragments will be produced
  8. Continue running cycles until Cycle 30; during this cycle over 1 billion target fragments will be produced
Name: Step 2
Name: Step 3
Name: Step 4
Name: Step 5
Name: Step 6
Name: Step 7




Research and Development

PCR - The Underlying Technology


What is the function of each component of a PCR reaction?

The template DNA is the original copy of DNA which is used as the variation in the experiment. Each set of template DNA should vary from organism to organism. Primers are short strands of DNA or RNA which bind to specific points on DNA. These primers are like beacons which show where replication should begin. Taq polymerase is a polymerase which operates under a specific temperature but can survive under extreme temperature variations. Deoxyribonucleotides are the individual building blocks that make up DNA.


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

First the DNA helix is separated by increasing the temperature to 95°C. After the helix is separated the DNA is cooled to 57°C so that the primers can attach to the single stranded DNA. The temperature is then raised to 72°C in order for the DNA polymerase to attach to the primers and then begin synthesizing the correct nucleotides onto the DNA. The polymerase continues till the entire strand is covered. This process is repeated until there is an abundance of the desired DNA fragment.


Which base anneals to each base listed below?

In DNA, adenine pairs with thymine and cytosine pairs with guanine.

During which two steps of thermal cycling does base-pairing occur? Explain your answers.

When primer attaches (Temp drops to 57℃) and when DNA polymerase is working (Temp is 72° C). The primer must match with the correct nucleotides. This shows that the bases are paired according to their nucleotide sequences. DNA polymerase must match the correct nucleotides with the existing DNA which also shows that the nucleotides must match.




SNP Information & Primer Design

Background: About the Disease SNP

The rs1044498 disease SNP, present in "Homo sapiens", is connected to bone disorders that are associated with type 2 diabetes mellitus (T2DM). Only one swap in the nucleic base causes this disease SNP. The one change in the nucleic base changes the codon which is read. The codon which is associated with our disease is CAG. The numerical position of the disease SNP is 131851228.



Primer Design and Testing

The diseased strand had one nucleotide that was changed. We then used the genome database and entered the two different sequences to determine which one was the diseased SNP.

Normal DNA Sequence
Diseased DNA Sequence