BME100 f2017:Group4 W1030 L4

BME 100 Fall 2017	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: Alexis Chavez Human Name: Jacob Schuler Slightly Above average student Name: Nazmina Ansari To Be Determined Name: Quentin Ellist The Man the Myth the Legend Name: Ryan Daughtry Famous Singer LAB 4 WRITE-UP Protocol Materials Lab Coat and disposable gloves PCR Reaction mix, 8 tubes, 50 microliters: Mix contains Taq DNA polymerase, MgCl2, and dNTP's DNA/Primer mix, 8 tubes, 50 microliters 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 Cup for discarded tips Micropipettor OpenPCR machine PCR Reaction Sample List Tube Label PCR Reaction Sample Patient ID G4 + Positive control G4 P G4 - Negative control G4 N G4 1-1 Patient 1, replicate 1 G4 1-1 (40178) G4 1-2 Patient 1, replicate 2 G4 1-2 (40178) G4 1-3 Patient 1, replicate 3 G4 1-3 (40178) G4 2-1 Patient 2, replicate 1 G4 2-1 (92225) G4 2-2 Patient 2, replicate 2 G4 2-2 (92225) G4 2-3 Patient 2, replicate 3 G4 2-3 (92225) DNA Sample Set-up Procedure Place the PCR reaction mix, consisting of Taq DNA Polymerase, Magnesium Chloride, and dNTP's, into the PCR tubes> Be sure to use disposable pipette tips, and replace them after ever use. Next, place the DNA/ primer mix into the PCR tubes, as these contain the different template DNA. Follow the same procedures regarding pipettes. Upon placing all of these together in the PCR tubes, place the tubes into the thermal cycler. OpenPCR program HEATED LID: 100 Degrees Celsius INITIAL STEP: 95 Degrees Celsius for 2 Minutes NUMBER OF CYCLES: 25 Denature at 95 Degrees Celsius for 30 seconds Anneal at 57 Degrees Celsius for 30 seconds Extend at 72 Degrees Celsius for 30 seconds FINAL STEP: 72 Degrees Celsius for 2 minutes FINAL HOLD: 4 Degrees Celsius Research and Development PCR - The Underlying Technology Template DNA Template DNA is the selected sequence of DNA typically between 75-1000 base pairs. It is the sequence being amplified by PCR Primers Primers are proteins that bind to very specific sequences within a gene. The primer then allow for a Polymerase (in the case Taq Polymerase) to bind to the primer and begin to copy the selected DNA sequence Taq Polymerase Taq Polymerase is a protein in the Polymerase family that allows for replication of a template strand of DNA. The reason Taq Polymerase is used instead of the body's natural DNA Polymerase is because Taq Polymerase can withstand the high temperatures required by PCR without denaturing. Taq Polymerase comes from the Thermus aquaticus bacteria. Deoxyribonucleotides Deoxyribonucleotides are special forms of Adenine, Guanine, Thymine, and Cytosine. The difference between these and standard nucleotides is that they are in their triphosphate form. This means that as Taq Polymerase adds these bases to the template strand the triphosphate breaks and provides energy for the polymerizing reaction. They are used to match up against the template strand of DNA and produce a complimentary strand. SNP Information & Primer Design Background: About the Disease SNP The disease SNP stands for Single nucleotide polymorphism. This genetic disease occurs when a specific variation in a single nucleotide of adenine, thymine, cytosine, or guanine. These nucleotides can be changed, removed, or added to a polynucleotide sequence. These changes can have a variety of effects upon the DNA of the organism, as it can cause drastic changes in the amino acid sequences. These can affect many things, like pathogenic responses, reactions to chemicals, medications, vaccinations, and the like. Primer Design and Testing The primers shown below were obtained by copying 20 nucleotides ending in the mutated nucleotide for the disease primer, and the normal nucleotide for the normal primer. The reverse primer was the same for both of these pairs. Diseased Pair Normal Pair