BME100 s2017:Group4 W1030AM L4: Difference between revisions
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# Label the empty PCR tubes with the appropriate descriptions. Two PCR tubes will be labeled as controls. The remaining 6 PCR tubes will be split into 2 sets of 3. One set will be the 3 replicates for patient 1 and will be labeled as such. The other set will be the 3 replicates for patient 2 and will be labeled as such. | # Label the empty PCR tubes with the appropriate descriptions. Two PCR tubes will be labeled as controls. The remaining 6 PCR tubes will be split into 2 sets of 3. One set will be the 3 replicates for patient 1 and will be labeled as such. The other set will be the 3 replicates for patient 2 and will be labeled as such. | ||
# Place the empty PCR tubes onto a PCR tube rack to secure them in an upright position | # Place the empty PCR tubes onto a PCR tube rack to secure them in an upright position | ||
# Use a micropipettor to move the pre-prepared 50μL PCR reaction mixes into their | # Use a micropipettor to move the pre-prepared 50μL PCR reaction mixes into their correspondingly labeled empty PCR tube. Make sure to use a new micropipettor tip each time. | ||
# Use a micropipettor to move the pre-prepared 50μL DNA/Primer mixes into their | # Use a micropipettor to move the pre-prepared 50μL DNA/Primer mixes into their correspondingly labeled PCR tube. Make sure to use a new micropipettor tip each time. | ||
# Place the PCR reaction mix tubes in the thermocycler. | # Place the PCR reaction mix tubes in the thermocycler. | ||
Revision as of 22:22, 21 March 2017
BME 100 Spring 2017 | 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
HEATED LID: 100°C INITIAL STEP: 95°C for 2 minutes to allow the DNA to denature and separate as much as possible NUMBER OF CYCLES: 25 Denature at 95°C for 30 seconds to allow the DNA to uncoil and the strands to separate Anneal at 57°C for 30 seconds to allow primers to attach to the DNA at a certain spot Extend at 72°C for 30 seconds to allow polymerase to extend the replicated DNA strand FINAL STEP: 72°C for 2 minutes to ensure that the DNA has been fully replicated FINAL HOLD: 4°C to let the DNA, in its fully replicated state, coil back together and act like DNA naturally would
Research and DevelopmentPCR - The Underlying Technology Q1: What is the function of each component of a PCR reaction?
Q2: What happens to the components (listed above) during each step of thermal cycling?
Q3: DNA is made up of four types of molecules called nucleotides, designated as A, T, C and G. Base-pairing, driven by hydrogen bonding, allows base pairs to stick together. Which base anneals to each base listed below? If you need help, use the “Build a DNA Molecule” tool at http://learn.genetics.utah.edu/content/begin/dna/builddna/
Q4: During which two steps of thermal cycling does base-pairing occur? Explain your answers Base-pairing occurs during annealing and extending. During annealing the Primers will anneal to the target DNA. Its bases will pair with the bases in the target DNA and has been designed to bind to a specific region of the DNA. During Extending, TAQ Polymerase will extend the primers by adding dNTPs to the target strand and make double stranded DNA. Extra Credit PCR Original Illustration
At around 57 degrees Celsius 2 primers anneal to the desired region in the template DNA. They attach to the 3' ends of the top and bottom strand because DNA polymerase always builds 5' to 3'.
At around 72 degrees Celsius, the DNA polymerase becomes active and attaches the complementary nucleotide bases until it reaches the end. Taq polymerase is used in particular because it operated well at high temperatures After elongation occurs, the whole denaturing, annealing, and elongating process repeats itself for around 25 cycles in this case. After the third cycle, DNA segments that are purely the desired regions begin to appear.
SNP Information & Primer DesignBackground: About the Disease SNP A SNP is a single nucleotide polymorphism, which means that one nucleotide is replaced with another. In the specific case of this disease SNP, an adenine nucleotide is replaced with a cytosine instead. The allele changes from AGT to CGT when this SNP occurs. Although the majority of SNPs do not have negative disease related consequences, this specific SNP is pathogenic. In particular, it is linked with Cystic Fibrosis and is found on chromosome 7:117587799 in Homo sapiens(humans).
General Background Information: What is a nucleotide? A nucleotide is a compound consisting of a nucleoside linked to a phosphate group. The nucleotides form the basic structural unit of nucleic acids such as DNA. What is a polymorphism? A polymorphism is the condition of multiple forms of a single gene.
Primer Design and Testing
There was no result for the disease positive allele. This is because it is a mutation, so it is not part of the normal human genome. While this mutation occurs, it does not happen to the average human, so it was unable to be found in the database.
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