BME100 s2018:Group1 W0800 L4

From OpenWetWare
Jump to navigationJump to search
Owwnotebook icon.png BME 100 Spring 2018 Home
People
Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
Course Logistics For Instructors
Photos
Wiki Editing Help
BME494 Asu logo.png

Our Team

Name: Carlie Rein
Role(s)
Name: Mikaela Hatfield
Role(s)
Name: Brandon Allred
Role(s)

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 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 groups


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G1 P Positive control none
G1 N Negative control none
G1 1-1 Patient 1, replicate 1 25053
G1 1-2 Patient 1, replicate 2 25053
G1 1-3 Patient 1, replicate 3 25053
G1 2-1 Patient 2, replicate 1 57814
G1 2-2 Patient 2, replicate 2 57814
G1 2-3 Patient 2, replicate 3 57814



DNA Sample Set-up Procedure

  1. Extract DNA sample from blood, saliva, hair, or other source
  2. Transfer DNA to PCR tube with micropipette (Reminder: Always replace the tips after every use)
  3. Add Primer 1 to PCR tube
  4. Add Primer 2 to PCR tube (This will attach to the second site)
  5. Add nucleotides to PCR tube
  6. Add TAQ DNA polymerase into PCR tube
  7. Close lid to tube and place in OpenPCR DNA Thermocycler


OpenPCR Program

  • Heated Lid: 100 degrees Celcius
  • Initial Step: 95 degrees Celcius for 2 minutes
  • Number of Cycles: 25
- Denature at 95 degrees Celcius for 30 seconds
- Anneal at 57 degrees Celcius for 30 seconds
- Extend at 72 degrees Celcius for 30 seconds
  • Final Step: 72 degrees Celcius for 2 minutes
  • Final Hold: 4 degrees Celcius

Research and Development

PCR - The Underlying Technology

Template DNA Provide the sequence to be copied
Primers Binds to the DNA in specific spots which will copy the desired portion of the DNA
Taq Polymerase Protein that starts the synthesis of new DNA strands, they read the DNA code and match it with appropriate nucleotides.
Deoxyribonucleotides (dNTP's) The nucleotides that bind to the DNA with base pairing which make up the new strands of DNA


Initial Step: 95 degrees celsius for 2 minutes This separates the template DNA double helix into two single stranded DNA molecules
Primers Binds to the DNA in specific spots which will copy the desired portion of the DNA
Denature at 95°C for 30 seconds: The bonds between continue DNA break and it separates into two separate strands
Anneal at 57°C for 30 seconds: The primers bind to DNA in two sites, marking which area will be copied
Extend at 72°C for 30 seconds: The polymerase binds to the primers and begins working to translate the DNA
FINAL STEP: 72°C for 2 minutes: Nucleotides are able to bind to DNA
FINAL HOLD: 4°C: Complete process

Adenine (A) pairs with Thymine (T) and vice versa Cytosine (C) pairs with Guanine (G) and vice versa

Q4. During which two steps of thermal cycling does base‐pairing occur? Explain your answers.

This occurs during the final step and the final hold because the DNA begins to be translated.

SNP Information and Primer Design

An investigation on DNA analysis was conducted on a gene named rs1044498. A nucleotides are the building blocks to a large structure called nucleic acid, which makes up DNA. Polymorphism is a term that explains genetic variance within a given population, and because of polymorphism, natural selection will select for more favorable genes and expressions. This gene variation is found in homo sapiens, and it is located on the sixth chromosome. The clinical significance of the single nucleotide polymorphism, or SNP, is with another allele and benign. The condition commonly associated with this condition is type 2 diabetes. EPP1 stands for Ectonucleotide Pyrophosphatase/Phosphodiesterase 1, and it served to bind in the 3’-5’ direction, as well as bind ATP and calcium ions. An allele is a pair of alternate forms of a gene that can be found on the same place on the chromosome and come to fruition as a result of mutation. The codon for this disease associated allele read CAG instead of AAG, and the numerical position of this codon is 131851228. The disease forward primer is as follows: GT TCA GAT GAC TGC AAG GAC C. The disease reverse primer is T GTT TAA AAG TTT CTT TAA TA.