BME100 s2018:Group1 W0800 L4

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
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 Extract DNA sample from blood, saliva, hair, or other source Transfer DNA to PCR tube with micropipette (Reminder: Always replace the tips after every use) Add Primer 1 to PCR tube Add Primer 2 to PCR tube (This will attach to the second site) Add nucleotides to PCR tube Add TAQ DNA polymerase into PCR tube 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.