BME100 f2013:W1200 Group5 L4: Difference between revisions
Line 89: | Line 89: | ||
'''PCR - The Underlying Technology'''<br> | '''PCR - The Underlying Technology'''<br> | ||
'''Components of a PCR Reaction'''<br> | '''Components of a PCR Reaction'''<br> | ||
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 | 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 you wish to amplify. 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. Therefore, to amplify the target strand of DNA, you need to have these nucleotides in a deoxyribonucleotide (dNTP) solution to pair to the existing strands of DNA. However, having the dNTP's is not enough. Primers are used as customizable units of complementary base pairs or nucleotides which can "seek out" and bind to the target sequence. This effectively creates starting point for the rest of the synthesis on the DNA strand. It is from this point that Taq Polymerase (a heat tolerant form of DNA polymerase) can bind and pull dNTP's out of the surrounding solution to match the target strand of DNA. However, Magnesium Chloride needs to be present in the solution to act as a catalyst for the reaction that Taq Polymerase carries out. | ||
'''PCR: The Process of Thermal Cycling'''<br> | '''PCR: The Process of Thermal Cycling'''<br> | ||
To initiate thermal cycling, the template DNA strand must be heated at a temperature of 95°C over 3 minutes. The heat will cause the double helix to straighten and then denature for 30 seconds. When the DNA strands denature they break apart at the nucleotides leaving each strand with its own set. | |||
[[Image:InitialStep.png]] | [[Image:InitialStep.png]] | ||
Revision as of 23:36, 29 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 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.
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. We started at 12:55pm and at 1:40 pm our machine had gone through 15 cycles.
ProtocolsPCR Protocol: The PCR machine begins by warming up to 100 degrees Celsius to begin the process. Then, the initial step takes place for three minutes at 95 degrees Celsius. This is done to heat up all the DNA strands, serving as a pre-denaturation step. Then the next three steps are repeated for a total of 35 cycles. The first step in this cycle is the denaturation cycle, and it is done at 95 degrees Celsius for 30 seconds. This is done to separate the double stranded DNA to single strands, forcing all reactions from enzymes to stop. The second step of the cycle is the annealing process, and it is done at 57 degrees Celsius for 30 seconds. This is when the polymerase attaches and begins to copy the DNA template. The last step of the cycle is the extending process, and it is done at 72 degrees Celsius for 30 seconds. This is when the polymerase couples to the primer on the 3' side, adding the bases that are complementary to the DNA template. The denaturation, annealing, and extension cycles are repeated for a total of 35 cycles. Then, the final step at 72 degrees Celsius for three minutes ensures that all the DNA goes through the extension process before the PCR machine cools down again. Finally, the PCR machine cools down to 4 degrees Celsius to ensure that all the single strands of DNA bond into double strands once again. DNA Sample Set-up
Research and DevelopmentPCR - The Underlying Technology Components of a PCR Reaction PCR: The Process of Thermal Cycling To initiate thermal cycling, the template DNA strand must be heated at a temperature of 95°C over 3 minutes. The heat will cause the double helix to straighten and then denature for 30 seconds. When the DNA strands denature they break apart at the nucleotides leaving each strand with its own set.
|