BME100 s2015:Group6 12pmL4

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: Justin Blommer Name: Ethan Mathew Name: Spencer Cobb Name: Sarah Jones Name: Abigail Rene LAB 4 WRITE-UP Protocol Materials Lab coat and disposable gloves PCR reaction mix, 8 tubes, 50μL each: Mix contains Taq DNA polymerase, MgCl2, and dNTP's DNA/primer mix, 8 tubes, 50μL 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 re-use disposable pipette tips or samples will be cross-contaminated Cup for discarded tips Micropipettor OpenPCR machine: shared by two groups PCR Reaction Sample List Tube Label PCR Reaction Sample Patient ID G6 P Positive control none G6 N Negative control none G6 1-1 Patient 1, replicate 1 11105 G6 1-2 Patient 1, replicate 2 11105 G6 1-3 Patient 1, replicate 3 11105 G6 2-1 Patient 2, replicate 1 22923 G6 2-2 Patient 2, replicate 2 22923 G6 2-3 Patient 2, replicate 3 22923 DNA Sample Set-up Procedure Step 1: Gather all materials: A PCR tube rack, 8 PCR tubes of 50 μL of PCR mix, 1 PCR tube of positive control, 1 PCR tube of negative control, 3 PCR tubes of Patient 1 DNA, 3 PCR tubes of Patient 2 DNA, PCR tube rack, micropipettor, micropipette tips, plastic cup, lab coat and gloves, and an OpenPCR machine. Step 2: Label the sides of the tubes with Group 6's modified tube labels and place them in the rack. Step 3: The 8 PCR tubes already containing the PCR reaction mix will be used in the the PCR machine. The provided DNA primer mixes will be pipetted into these 8 PCR reaction mix tubes. Step 4: Transfer 50 μL of positive control into tube #1. Step 5: Transfer 50 μL of negative control into tube #2. Step 6: Transfer 50 μL of Patient 1 (ID: 11105) DNA sample into tubes #3, #4, and #5. Step 7: Transfer 50 μL of Patient 2 (ID: 22923) DNA sample into tubes #6, #7, and #8. Step 8: Now, there are 100 μL of complete PCR reaction in each tube. Step 8: Ensure the lids are closed tight. Place the tubes in the PCR machine’s heating block and once all slots in the machine are filled, start the machine cycle. Note: Ensure to replace pipette tip with a clean one between every pipette. OpenPCR program Pictured above is the initial DNA strand before heat is applied The initial cycle requires the temperature to increase to 95°C for 2 minutes 1) The temperature is increased to 95°C for 30 seconds. The strands of DNA separate into single strands 2) The temperature is decreased to 57°C and primers bind to complimentary matches on the target DNA sequence 3) The temperature is increased to 72°C and taq polymerase binds to the primers and add nucleotides to extend the second strand Steps 1-3 are repeated for a total of 35 cycles Research and Development PCR - The Underlying Technology