BME100 s2015:Group10 12pmL4: Difference between revisions
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'''DNA Sample Set-up Procedure''' | '''DNA Sample Set-up Procedure''' | ||
<!-- In your own words, write an easy-to-comprehend list of the steps your group took to set up the PCR reaction in the PCR reaction tubes. End with placing the tubes in to the thermal cycler. Do not copy-paste the instructions from the Workbook. That will be considered plagiarism. --> | <!-- In your own words, write an easy-to-comprehend list of the steps your group took to set up the PCR reaction in the PCR reaction tubes. End with placing the tubes in to the thermal cycler. Do not copy-paste the instructions from the Workbook. That will be considered plagiarism. --> | ||
# Step 1 | # Step 1: Obtain the necessary equipment from the supervisor. | ||
# Step 2 | # Step 2: Label all your tubes indicating the patient and the trial number, and a positive and negative control. | ||
# Step 3... | # Step 3: Pipet the patient samples into the appropriate corresponding test tube. Make in each tube there is the DNA/Primer mix, and patient samples/control. The total volume should be 100ul in each tube. Be sure to not cross contaminate the samples with used tips. | ||
# Step 4: Once all your samples have the right mixes in each of them, place the tubes in the thermocycler. | |||
'''OpenPCR program''' | '''OpenPCR program''' | ||
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[[Image:PCR steps.jpg]] | [[Image:PCR steps group 10 PM.jpg]] | ||
[[Image:TEAM_10_PM_PCR.jpg]] | |||
Inspired by: http://users.ugent.be/~avierstr/principles/pcr.html | |||
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<!-- Add a write-up, essay-style, organized into paragraphs with descriptive headers, based on the questions and answers from the Research and Development exercise. 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. You are not allowed to use images from current or past BME 100 students' reports on OpenWetWare. --> | <!-- Add a write-up, essay-style, organized into paragraphs with descriptive headers, based on the questions and answers from the Research and Development exercise. 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. You are not allowed to use images from current or past BME 100 students' reports on OpenWetWare. --> | ||
'''Denaturation: The DNA strands are heated to a high temperature of about 95C. | '''Denaturation:''' Before the DNA strands are put into the Heated lid, the '''template strand, the strand which has the portion to be copied,''' is preheated to 100C. The DNA strands are then heated to a high temperature of about 95C. After thirty seconds the hydrogen bonds weaken which hold the double helix together, at which point the bonds become so weak, the strands physically separate. The '''dNTP's, or deoxyribonucleotide base pairs,''' will be exposed and the DNA is ready for primers to be attached. | ||
Annealing: At a lower temperature, of about | '''Annealing:''' At a lower temperature, of about 50C and the time has been another thirty seconds, the '''primers, the starting points for copying that assist enzymes,''' are introduced into the mix. Since the DNA base pairs are exposed, the primers will be able to bind to specifically selected sites on the DNA. After this portion, the replication will begin. | ||
Extension: DNA | '''Extension:''' '''Taq polymerase, a special kind of replicating enzyme for DNA,''' begins to line up the new base pairs at a temperature of 72C and after another thirty seconds has passed. At this period of time, the DNA will have been doubled. The previous three steps will be repeated 35 times in order to amplify a single section of the DNA to be copied millions and millions of times. | ||
Final Elongation: The DNA is put through one last extension to ensure all the strands have matching base pairs to form complete double helices. | '''Final Elongation:''' The DNA is put through one last extension to ensure all the strands have matching base pairs to form complete double helices. This is done by heating the DNA to 72C and this is done for 2 minutes. | ||
Final Hold: The DNA is stored | '''Final Hold:''' The DNA is stored at 4C to prevent it from denaturing again. | ||
Latest revision as of 23:50, 31 March 2015
BME 100 Spring 2015 | 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 4 WRITE-UPProtocolMaterials
OpenPCR program
Research and DevelopmentPCR - The Underlying Technology Denaturation: Before the DNA strands are put into the Heated lid, the template strand, the strand which has the portion to be copied, is preheated to 100C. The DNA strands are then heated to a high temperature of about 95C. After thirty seconds the hydrogen bonds weaken which hold the double helix together, at which point the bonds become so weak, the strands physically separate. The dNTP's, or deoxyribonucleotide base pairs, will be exposed and the DNA is ready for primers to be attached. Annealing: At a lower temperature, of about 50C and the time has been another thirty seconds, the primers, the starting points for copying that assist enzymes, are introduced into the mix. Since the DNA base pairs are exposed, the primers will be able to bind to specifically selected sites on the DNA. After this portion, the replication will begin. Extension: Taq polymerase, a special kind of replicating enzyme for DNA, begins to line up the new base pairs at a temperature of 72C and after another thirty seconds has passed. At this period of time, the DNA will have been doubled. The previous three steps will be repeated 35 times in order to amplify a single section of the DNA to be copied millions and millions of times. Final Elongation: The DNA is put through one last extension to ensure all the strands have matching base pairs to form complete double helices. This is done by heating the DNA to 72C and this is done for 2 minutes. Final Hold: The DNA is stored at 4C to prevent it from denaturing again.
Credit: http://en.wikipedia.org/wiki/Polymerase_chain_reaction#Procedure
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