BME100 f2014:Group4 L4

BME 100 Fall 2014	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: Shreya Ramkumar Name: Anyssa Iwamoto Name: Israel Ortiz Name: Zach Sledge Role(s) Name: Dominique Stewart Role(s) Name: Larrison Black Role(s) LAB 4 WRITE-UP Protocol Materials Lab coat and disposable gloves PCR reaction 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 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 G4 + Positive control none G4 - Negative control none G4 1-1 Patient 1, replicate 1 85470 G4 1-2 Patient 1, replicate 2 85470 G4 1-3 Patient 1, replicate 3 85470 G4 2-1 Patient 2, replicate 1 90113 G4 2-2 Patient 2, replicate 2 90113 G4 2-3 Patient 2, replicate 3 90113 DNA Sample Set-up Procedure Divide the 8 PCR tubes in half Label tubes according to DNA/primer mix (positive, negative, patient 1 replicate 1, patient 1 replicate 2, ...) Place tubes in the rack Change the setting on the micropippettor to 50μL Transfer 50μL of PCR reaction mix into the tube which was labeled as the positive control Dispose used pipette tip Insert new pipette tip Transfer 50μL of positive control DNA/primer mix into the tube labeled positive control. Close the lids of the tubes tightly Set-up the PCR machine according to the instructions located in the "Open PCR program" section. Place tubes into the slots in the heating block of the PCR machine When all 16 slots are filled (multiple groups use one PCR) run the program Wait until the conclusion of the PCR program Record all data Return reusable materials Dispose waste such as used tubes, gloves, pipette tips, etc. into the Biohazard Bag Clean table OpenPCR program The following information should be inputted into the PCR machine in order to set it up for the experiment according to the Manual: Heated Lid: 100°C Initial Step: 95°C for two 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 two minutes Final Hold: 4°C PCR Components There are three major components in a polymerase chain reaction. The first components needed are the primers which are short single stranded DNA sequences. These two primers are synthesized to match and copy the beginning and end of the DNA. The second component needed is the polymerase enzyme that reads the DNA code and builds a copy. The last component needed for the chain reaction is DNA so that the polymerase enzyme can copy. PCR Process There are three major steps involved in a polymerase chain reaction, and these steps were repeated 35 times to generate and create DNA copies exponentially. The first step is called Denaturation, which is when the double-stranded DNA melts and separates into two single-stranded pieces of DNA at 94° C. The second step is called Annealing and this occurs at temperatures around 54° C. During the Annealing step the primers pair up to the single-stranded DNA pieces and the polymerase enzyme attaches and starts the DNA code copying. The last step is the extension step and that occurs optimally at 72° C where the DNA template is coupled with the primer and creates a double-strand DNA string. The following images are from the Michael McPherson & Simon Moller “PCR: The Basics” book.