BME100 s2015:Group2 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 Ryan Bridges Role(s) Name: Michael Bejarano Role(s) Name: Virginia Fernandez Role(s) Morgan Johnson 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 (http://www.promega.com/resources/protocols/product-information-sheets/g/gotaqcolorless-master-mix-m714-protocol/) 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 G2 + Positive control none G2 - Negative control none G2 1-1 Patient 1, replicate 1 15062 G2 1-2 Patient 1, replicate 2 15062 G2 1-3 Patient 1, replicate 3 15062 G2 2-1 Patient 2, replicate 1 95748 G2 2-2 Patient 2, replicate 2 95748 G2 2-3 Patient 2, replicate 3 95748 DNA Sample Set-up Procedure Step 1 Obtain all list materials above Step 2 The PCR tubes will need to be in strips of four, cut the initial strip of eight into a strip of four; OpenPCR machine only accepts strips of four or less. Step 3 WARNING label the tube with a TUBE LABEL, and ONLY WRITE on the LABELS; DO NOT WRITE on the TUBES. Step 4 Once tubes are correctly set-up place them in the rack Step 5 Label one of the tube as Positive Control Step 6 Label an additional tube as Negative Control Step 7 Set the pipette to transfer 50-MircoLiters Step 8 Attach a disposable pipette tip to the pipette Step 9 Using the pipette draw up 50-MircoLiters of PCR reaction mixture, and dispense the PCR mixture into the Positive Control tube. Step 10 Discard the disposable tip in the cup, after each use. Disposable tips are SINGLE USE ONLY, only one disposable tip per draw. i.e. draw-up PCR reaction mixture ten times it will require ten disposable tips. AVOID CONTAMINATION of the samples Step 11 Using the pipette, with a new disposable tip (single use only), draw up 50-MircoLiters of the Positive Control DNA/Primer Mix, and transfer it into the Positive Control tube. Step 12 Close the PCR tubes for Positive Control TIGHTLY Step 13 repeat steps 8-12 for NEGATIVE CONTROL now Step 14 Bring you tubes to TA at the PCR Machine, and with oversight set the PCR Machine as listed in OpenPCR Program. Step 15 With the APPROVAL of the TA place you tubes into the PCR Machine, and record the location of your tubes within the PCR Machine i.e. row & column. 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 Research and Development PCR - The Underlying Technology Q1. What is the function of each component of a PCR reaction? Template DNA: contains the sequence of DNA you wanted to amplify. Primers: short strands of DNA that adhere to the target segment. They identify the portion of DNA to be multiplied and provide a starting place for replication. Taq Polymerase: the enzyme that is in charge of replicating DNA. This is the polymerase part of the name polymerase chain reaction. Deoxyribonucleotides (dNTP’s): are needed so the DNA polymerase has building blocks to work with. Q2. What happens to the components (listed above) during each step of thermal cycling? INITIAL STEP: 95°C for 3 minutes: In order for the strand to denature completely, it is thoroughly heated to 95°C. Denature at 95°C for 30 seconds: The alpha-helix and beta sheets in a protein are obstructed and bonding between the secondary and tertiary structures disrupted, resulting in the separation of the strands. Anneal at 57°C for 30 seconds: A primer is annealed to each single strand of DNA in specific regions. Extend at 72°C for 30 seconds: Each primer is extended in the 5' to 3' direction to duplicate the specific regions of DNA to which the primer is attached. FINAL STEP: 72°C for 3 minutes: The product produced by the previous step, a nucleotide, attaches to the primer it is complementary to. FINAL HOLD: 4°C: Four strands of DNA are left to be cooled and Taq polymerase is deactivated. Q3. DNA is made up of four types of molecules called nucleotides, designated as A, T, C and G. Base-pairing, driven by hydrogen bonding, allows base pairs to stick together. Which base anneals to each base listed below? Adenine (A): Thymine Thymine (T): Adenine Cytosine (C): Guanine Guanine (G): Cytosine Q4. During which two steps of thermal cycling does base-pairing occur?