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Revision as of 12:17, 27 October 2013
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 TEAMLAB 1 WRITE-UPInitial Machine TestingThe Original Design
When we unplugged (part 3) from (part 6), the machine ... (did what? fill in your answer) When we unplugged the white wire that connects (part 6) to (part 2), the machine ... (did what? fill in your answer)
(Write the date you first tested Open PCR and your experience(s) with the machine)
ProtocolsThermal Cycler Program
Research and DevelopmentPCR - The Underlying Technology
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. |