BME100 f2013:W1200 Group14 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 TEAMLAB 1 WRITE-UPInitial Machine TestingThe Original Design Experimenting With the Connections 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)
ProtocolsDNA Sample Set-up
Materials
1. 1 Cycle at 95°C for 3 minutes PCR Reaction Mix
Research and DevelopmentPCR - 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) PCR, or polymerase chain reaction, is used to amplify DNA to diagnose disease, map the human genome, or even to solve crimes. There are several components involved in completing PCR-- the template DNA, the primers, the Taq polymerase, magnesium chloride, and deoxyribonucleotides. The purpose of each component is as follows: Template DNA: The template DNA contains the target sequence, and is the basis for the amplification. Without the template, the target sequence cannot be amplified, and the primers have nothing to bind to, and polymerase has nothing to copy. Primers: The primers bind to each single-stranded DNA and functions almost as a signal for Taq polymerase to begin the construction of the complementary sequence. Furthermore, the presence of both forward and reverse primers ensures that the target sequence, in particular, will be amplified, because the primers are made specifically for the sections of the template DNA which include the target sequence. Taq polymerase: This is an enzyme which constructs the complimentary sequence to the single strands of the template DNA. Magnesium chloride: The magnesium chloride acts as a catylyst for the polymerase reaction, and helps the reaction move along faster. Deoxyribonucleotides: These are free-floating nucleotides, and these are the bases that are used by Taq polymerase to create the complimentary sequence for the single-stranded DNA. The aforementioned materials are the key components in a PCR reaction. The first step of PCR is to heat the DNA to around 95 degrees celsius for around 3 minutes, and this allows the DNA to denature and unwind into two separate strands. The temperature, which is near boiling, and the required time, are both imperative to this reaction because this is the amount of energy that is required to break the hydrogen bonds between the nucleotides of the complementary strands. Once the DNA denatures, the temperature must be lowered to 57 degrees for around 30 seconds. The lowering of temperature allows for the forward and reverse primers to bind onto each single stranded-DNA. After 30 seconds of this, the temperature is once again re-heated to 72 degrees, the ideal temperature for Taq polymerase. Taq polymerase now begins constructing complementary strands to each single template strand. This continues for around 3 minutes. This cycle repeats itself over and over, until the "target sequence" is amplified more so than the original template DNA. The strands are complementary, and are created with paired nucleotides-- adenine binds with thymine, and cytosine with guanine. Base pairing, which is done through hydrogen bonds, are what create the "spiral staircase" of DNA structure. This is also what allows primers to the template 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.)
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