BME100 f2014:Group28 L4

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OUR TEAM Name: Andrew W. Hamidy Name: Mohammed Alhusayni Name: Brandt Hansen Name: Kyle Brague Name: Diego E. Reyes Name: Kyle Henriksen 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 OpenPCR machine: shared by two groups PCR Reaction Sample List Tube Label PCR Reaction Sample Patient ID G28 + Positive control 76447 G28 - Negative control 58040 G28 1-1 Patient 1, replicate 1 G28 1-2 Patient 1, replicate 2 G28 1-3 Patient 1, replicate 3 G28 2-1 Patient 2, replicate 1 G28 2-2 Patient 2, replicate 2 G28 2-3 Patient 2, replicate 3 DNA Sample Set-up Procedure Get the patient samples as well as the positive and negative controls from TA. Do not open the tubes just yet. Each set of samples has a unique number associated with it known as the patient ID number. There should be three replicates of each sample. Create labels that will be used to distinguish tubes. For example G51P, or G51N, G51 1-1. Etc. Make sure to record a key to each tube for future reference. 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 sec FINAL STEP: 72°C for 2 minutes FINAL HOLD: 4°C Research and Development PCR - The Underlying Technology Function of Components Primers- Pieces of DNA constructed in a lab to specifically locate a certain sequence of DNA in which is to be copied. These primers are usually 20 nucleotides long to locate the desired sequence in DNA. Primase- In nature, this is what starts off PCR instead of lab created primers. DNA Polymerase- Is a naturally occurring complex of proteins that attaches to the DNA and copies it with nucleotides before it is split into two. When used with a primer, it attaches itself to the end of the primer and starts to add nucleotides to complete the double strand of DNA. Nucleotides- Building blocks of DNA that include Adenine, Cytosine, Guanine, and Thymine (A,C,G,T). These nucleotides are grabbed by the DNA Polymerase and attached to the primer which completes the DNA strand. This information was taken from http://learn.genetics.utah.edu/content/labs/pcr/* What happens to the components (listed above) during each step of thermal cycling? INITIAL STEP: 95°C for 3 minutes: The DNA starts to lose it's double helix structure, becoming closer to a ladder. Denature at 95°C for 30 seconds: The DNA separates into two strands. Anneal at 57°C for 30 seconds: Primers bind to complementary matches on the target sequence Extend at 72°C for 30 seconds: Taq polymerase binds to the primed sequences and adds nucleotides to the strand. FINALSTEP: 72°C for 3 minutes: New sets of DNA are made swiftly without destruction of the double helix FINAL HOLD: 4°C: Allows the machine to shut off completely and complete the reaction This information was taken from http://openpcr.org/what-is-pcr Adenine anneals to thymine. Thymine anneals to adenine. Guanine anneals to cytosine. Cytosine anneals to guanine. This information was taken from http://learn.genetics.utah.edu/content/labs/pcr/*