BME100 s2015:Group1 9amL4

BME 100 Spring 2015	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: Grace Kim Name: Jonathan Moreno Name: Shawn Striker Name: student Role(s) Name: student Role(s) Name: student Role(s) LAB 4 WRITE-UP Protocol Materials Lab coat Disposable gloves PCR reaction mix DNA/primer mix 16 tubes (50 micro-liters) Strip of empty PCR tubes Disposable pipette tips Cup for discarded pipette tips Micropipettor OpenPCR machine PCR Reaction Sample List Tube Label PCR Reaction Sample Patient ID G1 + Positive control none G1 - Negative control none G1 1-1 Patient 1, replicate 1 11316 G1 1-2 Patient 1, replicate 2 11316 G1 1-3 Patient 1, replicate 3 11316 G1 2-1 Patient 2, replicate 1 25353 G1 2-2 Patient 2, replicate 2 25353 G1 2-3 Patient 2, replicate 3 25353 DNA Sample Set-up Procedure Gather materials Set aside patients' DNA samples and PCR reaction mix Cut the strip of empty PCR tubes to create two strips of four linked tubes Label the sides of the empty tubes appropriately Place the PCR tubes in a rack Place a new pipette tip onto the micropipettor Extract 50 μL of PCR reaction mix Release PCR reaction mix into corresponding PCR tube Discard pipette tip into cup Place a new pipette tip onto the micropipettot Extract 50 μL of DNA/primer mix Release DNA/primer mix into the PCR tube used in Step 8 Repeat Steps 6-12 for the rest of the tubes Close lids tightly Place the tubes in the PCR machine OpenPCR program Heated Lid: 100°C Initial Step: 95°C for 2 minutes Number of Cycles: 35 -Denature at 95°C for 30 seconds -Anneal at 57°C for 30 seconds -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 Q1. What is the function of each component of a PCR reaction? Template DNA: desired segment of the DNA intended for amplification Primars: attach to either end of the DNA segment that is being replicated Taq Polymerase: reads DNA code and than attaches matching nucleotides to complete the DNA replication dNTP's: building blocks of DNA Q2. What happens to the components (listed above) during each step of thermal cycling? Initial Step (95 degrees celsius): the DNA strand is Heated to 95 degrees celsius Denature (95 degrees celsius): DNA separated into two single DNA strands Anneal (57 degrees celsius): Primer connects to designated targets before DNA strands pair back together Extend (72 degree celsius): triggers the DNA polymerase which locates and connects to the primer on the DNA strand at either end Final Step (72 degrees celsius): complementary nucleotides are added to the strand Final Hold (4 degrees celsius): Taq polymerase is deactivated. 4 separate strands of DNA are left and the reaction takes place again to make two complete DNA strands, and so forth Q3. Which base anneals to each base listed below? Adenine (A): T Thymine (T): A Cytosine (C): G Guanine (G): C Q4. During which two steps of thermal cycling does base-pairing occur? Extended and Final Step. Taq polymerase is at the optimal temperature of 72 degrees celsius and can attach to primers on template DNA strands. Corresponding base pairs are then gathered to create a complementary DNA strand for the amplification process.