BME100 f2018:Group13 T1030 L4

BME 100 Fall 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:McKenna Godman student Role(s) Name:Haley Ellis student Role(s) Name:Amanda Hackett student Role(s) Name:Ali Fahy student Role(s) Name:Jacob Ries student Role(s) LAB 4 WRITE-UP Protocol Materials Lab Coat and disposable gloves PCR reacon mix, 8 tubes, 50 μL each: Mix contains Taq DNA polymerase, MgCl 2, 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 pipettes ps: 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 G13 + Positive control none G13 - Negative control none G13 1-1 Patient 1, replicate 1 96033 G13 1-2 Patient 1, replicate 2 96033 G13 1-3 Patient 1, replicate 3 96033 G13 2-1 Patient 2, replicate 1 38209 G13 2-2 Patient 2, replicate 2 38209 G13 2-3 Patient 2, replicate 3 38209 DNA Sample Set-up Procedure Step 1 Step 2 Step 3... 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 30 seconds FINAL STEP: 72°C for 2 minutes FINAL HOLD: 4°C Research and Development PCR - The Underlying Technology Template DNA in a PCR reaction functions as the base point for the reaction, it is what is being copied. The primers target specific sites on the DNA strands in order to copy specific DNA sequences. Taq polymerase attaches where the primer ends, and starts copying the DNA strand. Deoxyribonucleotides(dNTP's) are the building blocks of DNA (A, C, G, T) that are being copied by the polymerase. After two minutes at 95 degrees Celsius the double helix of the template DNA separates into two single-stranded DNA molecules, this will take about thirty seconds. Then the thermal cycler cools to 57 degrees, and after thirty seconds primers will attach to single-stranded DNA molecules. Once the temperature is raised to 72 degrees Celsius, the Taq polymerase is activated, and begins to add nucleotides onto each single strand until the process is complete (approximately two minutes). The correct nucleotide pairings are A with T and G with C, and vice versa. The two steps in the cycle where base-pairing occurs are during Anneal and Extend. At the Anneal step, the DNA has just separated, so the single-stranded DNA is looking for something to pair with, and this is when the primer comes in, to prevent the two strands from rejoining. During the Extend step, the single-stranded DNA is able to pair with other nucleotides because of the polymerase. SNP Information & Primer Design Background: About the Disease SNP SNP variation is found in homo sapiens on chromosome 12, also known as LRRK2 (leucine rich repeat kinase 2). The chromosome is responsible for ATP Binding, GTP Binding, and GTP-dependent protein kinase activity. When alleles of this gene are mutated, the healthy GTG codon found at position 40315266 is replaced with a GAG codon, resulting in SNP disease. While its clinical significance is uncertain, SNP is linked to familial parkinsonism. Primer Design and Testing PCR reactions require primers to amplify DNA. After designing forward and reverse primers for both the diseased and non-diseased gene codon, they may be validated using the non-disease human genome sequence. The non-disease primers designed for this PCR reaction yielded results 220 bp sequences away from the chromosome being analyzed. The primers are viable.