BME100 f2013:W1200 Group11 L4: Difference between revisions
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Polymerase Chain Reactions require several components to ensure the effective amplification of the target DNA. | Polymerase Chain Reactions require several components to ensure the effective amplification of the target DNA. | ||
For PCR to occur, template DNA is necessary. Template DNA is the sample DNA used in the PCR reaction that contains | For PCR to occur, template DNA is necessary. Template DNA is the sample DNA used in the PCR reaction that contains | ||
the target region, or section of DNA that | the target region, or section of DNA that can be amplified. When the template DNA is denatured, it is separated into two strands. | ||
Each strand is used as a template for the construction of a complementary DNA strand. Another component of a PCR reaction is primers. | Each strand is used as a template for the construction of a complementary DNA strand. Another component of a PCR reaction is primers. | ||
Primers are short pieces of single-stranded DNA that are complementary to a section of the template strand. In order to ensure the | Primers are short pieces of single-stranded DNA that are complementary to a section of the template strand. In order to ensure the | ||
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the DNA strand from the 5' to the 3' direction, while the reverse primer initiates the elongation of the DNA strand from the 3' to the 5' direction. | the DNA strand from the 5' to the 3' direction, while the reverse primer initiates the elongation of the DNA strand from the 3' to the 5' direction. | ||
In this way, the primers begin the construction of complementary DNA strands to each strand of the template DNA at the location of the target DNA. | In this way, the primers begin the construction of complementary DNA strands to each strand of the template DNA at the location of the target DNA. | ||
Taq polymerase | Taq polymerase start the production of new strands of DNA by connecting to dNTP (deoxyribonucleotides triphosphate) to match with the target sequence | ||
of the DNA. Magnesium Chloride (MgCl2) is the polymerase that needs a divalent cation to function correctly. This bonds assists by being a cofactor when | of the DNA. Magnesium Chloride (MgCl2) is the polymerase that needs a divalent cation to function correctly. This bonds assists by being a cofactor when | ||
the polymerase and the DNA strand bind together. The polymerase with the hydroxide group is required for the bind and helps in removing the hydrogen from the | the polymerase and the DNA strand bind together. The polymerase with the hydroxide group is required for the bind and helps in removing the hydrogen from the | ||
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[[Image:121314.jpg]]<br>After the forward and reverse primers have bound to separate strands of the DNA template, the DNA polymerase is ready to synthesize new DNA strands complementary to the template strands. <br> | [[Image:121314.jpg]]<br>After the forward and reverse primers have bound to separate strands of the DNA template, the DNA polymerase is ready to synthesize new DNA strands complementary to the template strands. <br> | ||
[[Image:121315.jpg]]<br>After the DNA polymerase has bound to the primer-template hybrid on each strand, it begins to create new strands using dNTPs as building blocks. After this occurs once, the original template strands and the newly synthesized complementary strands each separate from each other during a second cycle of denaturation. Then, forward and reverse primers anneal again, the DNA polymerase attaches once more, and complementary strands are synthesized. After multiple cycles of denaturation, annealing, and extension, the DNA polymerase is responsible for the amplification of the target region. <br> | [[Image:121315.jpg]]<br>After the DNA polymerase has bound to the primer-template hybrid on each strand, it begins to create new strands using dNTPs as building blocks. After this occurs once, the original template strands and the newly synthesized complementary strands each separate from each other during a second cycle of denaturation. Then, forward and reverse primers anneal again, the DNA polymerase attaches once more, and complementary strands are synthesized. After multiple cycles of denaturation, annealing, and extension, the DNA polymerase is responsible for the amplification of the target region. <br> | ||
<br><br> | <br><br> | ||
Revision as of 18:08, 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 (part 3) from (part 6), the LCD screen on the machine remained lit, but did not display any information on the screen. When we unplugged the white wire that connects (part 6) to (part 2), the machine
We first tested the Open PCR machine on October 23, 2013. During this test run we had unsuccessful result, as our Open PCR machine failed to operate. ProtocolsThermal Cycler Program 1. Denature for one cycle at 95C for three minutes (Initial hold)
3. Extend the DNA at 72C for 3 minutes (to stabilize the DNA) DNA Sample Set-up
DNA Sample Set-up Procedure
Research and Development'PCR - The Underlying Technology' 'Components of a PCR Reaction'
'Steps of Thermal Cycling'
Thermal cycling initial starts at 95°C for three minutes during this period of time the enzymes are activated due to the optimal temperature that taq
polymerase will become active. During this step the single stranded DNA template will also begin to disconnect. The next step is Denature which will be at
95°C for 30 second throughout this time period the hydrogen bonds with the complementary bases will begin to become distorted ultimately causing the DNA
melting of the DNA template producing a single-stranded DNA molecule.
Following denaturation, the DNA undergoes annealing, where primers form stable DNA-DNA hydrogen bonds with the template DNA. After the template DNA
and the primers have bound, the DNA polymerase attaches to the primer-template hybrid. Annealing occurs at 57 degrees Celsius for 30 seconds.
Then, extension occurs at 72 degrees Celsius for 30 seconds. During extension, the DNA polymerase enzyme synthesizes a new strand of DNA complementary
to the template strand by adding dNTPs complementary to the bases of the template strand in the 5' to 3' direction.
During the final step which occurs at 72 degrees Celsius for duration of three minutes any remaining single stranded DNA that is leftover will become fully extended.
Also during the final step, the final hold occurs at four degree Celsius, which is when the entire reaction becomes stored.
'Diagram of PCR'
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