The Original Design
The Open PCR machine is a machine that uses heat cycles to replicate specific DNA strands. DNA is placed in PCR tubes, which are placed in the PCR block. The machine, which is hooked up to a computer, is programmed to cycle through different temperatures. The heated lid is set to 100°C and the machine is initially set to 95°C for three minutes. The machine then goes through 35 cycles of 95°C for 30 seconds (which splits the DNA into two strands), 57°C for 30 seconds (which is when primers bind to the target sequences and begin to replicate the strand) and then 72°C for 30 seconds (which by the end has created double the number of DNA strands each cycle started with). It is then set at 72°C for three minutes and then a final hold temperature of 4°C.
Experimenting With the Connections
When we unplugged the screen from the mother board, the screen on the machine turned off.
When we unplugged the white wire that connects the mother board to the PCR block, the temperature reading on the machine became incorrect and read that it was at -40°C.
Test Run
On October 23, 2013 we tested the Open PCR machine and found that it worked well. The temperatures fluctuated somewhat when it went through the heat cycles, but usually only by about .1°-.2°. There was somewhat of a lag between the readings on the machine and the readings on the computer as well. As for the timing of the cycles, the machine worked correctly and cycled through the temperatures at the correct time.
Protocols
Thermal Cycler Program
DNA Sample Set-up
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DNA Sample Set-up Procedure
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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
(Add a write-up, essay-style, organized into paragrpahs with descriptive headers, based on the Q&A's from Section three of your worksheet) Components of a PCR Reaction
A PCR reaction needs many components to synthesize a strand of DNA including, template DNA, primers, Taq Polymerase, Magnesium Chloride (MgCl2), and Deoxyribonucleotides. The template strand of DNA is the original strand of DNA that contains the target sequence that you wish to amplfy. DNA is made of up the nucleotides Adenine (A), Thymine(T), Cytosine (C), and Guanine(G) which pair in a A-T or C-G format. Primers are used to isolate the target target sequence because they can be customized to contain the compelentary base pairs (or nucleotides) of the sequence. They can effectively "seek out" the target sequence on both strands of the template DNA and bind, creating a starting point for synthesis.
PCR: The Process of Thermal Cycling
(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.)
BME 100 Fall 2013
