BME100 f2013:W1200 Group10 L4

BME 100 Fall 2013	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: Robert Childers Role(s) Name: Akshara Malla Role(s) Name: Daniel Palmer Role(s) Name: student Role(s) Name: student Role(s) Name: Jordan Jones Role(s) LAB 1 WRITE-UP Initial Machine Testing The Original Design (Add image of the full OpenPCR machine here, from the Week 9 exercise. Write a paragraph description for visitors who have no idea what this is) Experimenting With the Connections When we unplugged (part 3) from (part 6), the machine's power to the LED screen was turned off. When we unplugged the white wire that connects (part 6) to (part 2), the sensors on the heat plate were disconnected. They were registering the temperature at -40 degrees Celsius until plugged back in. Test Run (Write the date you first tested Open PCR and your experience(s) with the machine) Protocols Thermal Cycler Program DNA Sample Set-up row 1 cell 1 row 1 cell 2 row 1 cell 3 row 1 cell 4 row 2 cell 1 row 2 cell 2 row 2 cell 3 row 2 cell 4 DNA Sample Set-up Procedure Step 1 Step 2 Step 3... PCR Reaction Mix What is in the PCR reaction mix? DNA/ primer mix What is in the DNA/ primer mix? Research and Development PCR - The Underlying Technology Polymerase Chain Reaction copies strands of DNA and amplifies with the presence of template DNA, primers, Taq Polymerase, Magnesium Chloride, and deoxyribonucleotides. DNA is a double stranded helix composed of a phosphate sugar (deoxyribose sugars) backbone and any of the four nitrogenous bases that include the purines (adenine and guanine) and the pyrimidines (cytosine and thymine). In the double helix, adenine and thymine form hydrogen bonds with each other while cytosine and guanine form the other base pair. The two strands are held together by hydrogen bonds between the nitrogenous bases. In the PCR reaction, the template DNA strand serves as the template of DNA which is to be copied by various enzymes. Enzymes read the template stand and compose a complementary strand based on the sequence of the template DNA. RNA Primase adds primers which are short strand of RNA to the DNA strands in order for DNA Polymerase to attach dNTPS as DNA Polymerase can only add nucleotides to an existing strand. Primers indicate the position where copying must begin on the DNA strand. The addition of primers also prevents the two DNA strands from rejoining. Taq Polymerase is a protein complex that copies DNA by adding dNTP's to the position on the strand as indicated by the primers. In order for DNA Polymerase to add dNTP's to the the DNA strand, magnesium chloride is necessary to catalyze the enzyme. Deoxyribonucleotides are unpaired nucleotides consisting of a phosphate, deoxyribose sugar, and a nitrogenous base that are used by DNA Polymerase to create the new complementary strand. (BONUS points: Use a program like Powerpoint, Word, Illustrator, Microsoft Paint, etc. to illustrate how primers bind to the cancer DNA template, and how Taq polymerases amplify the DNA. Screen-captures from the PCR video/ tutorial might be useful. Be sure to credit the sources if you borrow images.)) The Polymerase Chain Reaction goes through thermal cycling to copy DNA. First the temperature is raised to 95°C for 30 seconds. At this temperature denaturing of DNA occurs. The hydrogen bonds holding the nucleotides together break apart, and the the double helix of DNA is effectively split in half. Next, the temperature is lowered to 57°C for another 30 seconds. This is the temperature at which DNA naturally attempts to rejoin. Instead, specific sequences of nucleotide are attached to by the lab made primers. This prevents the DNA from rejoining, and indicates to the polymerase where to begin copying. In the final step of the cycle, the temperature is then increased to 72°C. At this temperature, the DNA polymerase begins to attach the excess dNTPs to the complementary pairing on the DNA. This process is catalyzed by the magnesium chloride in the solution. After 3 cycles, the desired dna sequence begins to form itself. These are indicated by strands of DNA with primers on both ends. The number of desired strands increases exponentially with each further cycle. After 30 cycles, there are 1 billion target strands. The solution is now effectively a pure solution of the target sequence.