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: student Role(s) Name: student Role(s) Name: student 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 Step 1 Step 2 Step 3... 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, and Extend at 72°C for 30 seconds FINAL STEP: 72°C for 2 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.