BME100 s2015:Group5 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: Jacob Licko Name: Stephen Belikoff Name: Khalil Khoury Name: Daniela Romero Name: Wei Lu 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/gotaq-colorless-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 Open PCR machine: shared by two groups PCR Reaction Sample List Tube Label PCR Reaction Sample Patient ID G5 P + Positive control none G5 N - Negative control none G5 1-1 Patient 1, replicate 1 59697 G5 1-2 Patient 1, replicate 2 59797 G5 1-3 Patient 1, replicate 3 59797 G5 2-1 Patient 2, replicate 1 79049 G5 2-2 Patient 2, replicate 2 79049 G5 2-3 Patient 2, replicate 3 79049 DNA Sample Set-up Procedure Gather all required materials (listed above) Divide strip of 8 empty tubes into two sets of four Follow the instructions from the Student Workbook and label each of the 8 tubes legibly with a black fine point marker in the following way: G (Group) # (Group Number) Patient # (1 or 2) [DASH] Sample # (1, 2, or 3) ((Two of the tubes must be labelled as "control." [P or N])) Patient Example: G5 1-2 (Group 5, Patient 1, Sample 2) Control Example: G5 P (Group 5, Positive Control) 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: This is the sequence of DNA that is intended for amplification. It will undergo extreme heat in order for it to denature. Primers: A primer is a single strand of DNA that serves as the starting point for DNA synthesis that binds to specific DNA sequences. Taq Polymerase: This is a thermostable enzyme that recombines nucleotides following separation by heat. It creates a new, complimentary strand of DNA through adding new nucleotides to the old ones. Deoxyribonucleotides (dNTP's): There are the building blocks of DNA: adenine (A), thymine (T), cytosine (C) and guanine (G). These are used by the Taq Polymerase to synthesize the complementary strand of DNA. Q2: What happens to the components (listed above) during each step of thermal cycling? 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): pairs with Thymine (T) Thymine (T): pairs with Adenine (A) Cytosine (C): pairs with Guanine (G) Guanine (G): pairs with Cytosine (C) Q4: During which two steps of thermal cycling does base-pairing occur? Explain your answers.