BME100 s2015:Group2 9amL4: 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. --> | ||
# | # Obtain all list materials above | ||
# | # The PCR tubes will need to be in strips of four, cut the initial strip of eight into a strip of four; OpenPCR machine only accepts strips of four or less. | ||
# | # WARNING label the tube with a TUBE LABEL, and ONLY WRITE on the LABELS; DO NOT WRITE on the TUBES. | ||
# Once tubes are correctly set-up place them in the rack | |||
# Label one of the tube as Positive Control | |||
# Label an additional tube as Negative Control | |||
# Set the pipette to transfer 50-MircoLiters | |||
# Attach a disposable pipette tip to the pipette | |||
# Using the pipette draw up 50-MircoLiters of PCR reaction mixture, and dispense the PCR mixture into the Positive Control tube. | |||
# Discard the disposable tip in the cup, after each use. Disposable tips are SINGLE USE ONLY, only one disposable tip per draw. i.e. draw-up PCR reaction mixture ten times it will require ten disposable tips. AVOID CONTAMINATION of the samples | |||
# Using the pipette, with a new disposable tip (single use only), draw up 50-MircoLiters of the Positive Control DNA/Primer Mix, and transfer it into the Positive Control tube. | |||
# Close the PCR tubes for Positive Control TIGHTLY | |||
# repeat steps 8-12 for NEGATIVE CONTROL now | |||
# Bring you tubes to TA at the PCR Machine, and with oversight set the PCR Machine as listed in OpenPCR Program. | |||
# With the APPROVAL of the TA place you tubes into the PCR Machine, and record the location of your tubes within the PCR Machine i.e. row & column. | |||
'''OpenPCR program''' | '''OpenPCR program''' | ||
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*HEATED LID: 100°C | *HEATED LID: 100°C | ||
*INITIAL STEP: 95°C for 2 minutes | |||
*NUMBER OF CYCLES: 35 | |||
Denature at 95°C for 30 seconds, Anneal at 57°C for 30 seconds, and *Extend at 72°C for 30 | |||
seconds | |||
*FINAL STEP: 72°C for 2 minutes | |||
*FINAL HOLD: 4°C | |||
<br><br> | |||
==Research and Development== | |||
'''PCR - The Underlying Technology'''<br> | |||
<!-- 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. --> | |||
Q1. What is the function of each component of a PCR reaction? | |||
*Template DNA: contains the sequence of DNA you wanted to amplify. | |||
*Primers: short strands of DNA that adhere to the target segment. They identify the portion of DNA to be multiplied and provide a starting place for replication. | |||
*Taq Polymerase: the enzyme that is in charge of replicating DNA. This is the polymerase part of the name polymerase chain reaction. | |||
*Deoxyribonucleotides (dNTP’s): are needed so the DNA polymerase has building blocks to work with. | |||
Q2. What happens to the components (listed above) during each step of thermal cycling? | |||
*INITIAL STEP: 95°C for 3 minutes: In order for the strand to denature completely, it is thoroughly heated to 95°C. | |||
*Denature at 95°C for 30 seconds: The alpha-helix and beta sheets in a protein are obstructed and bonding between the secondary and tertiary structures disrupted, resulting in the separation of the strands. | |||
*Anneal at 57°C for 30 seconds: A primer is annealed to each single strand of DNA in specific regions. | |||
*Extend at 72°C for 30 seconds: Each primer is extended in the 5' to 3' direction to duplicate the specific regions of DNA to which the primer is attached. | |||
*FINAL STEP: 72°C for 3 minutes: The product produced by the previous step, a nucleotide, attaches to the primer it is complementary to. | |||
*FINAL HOLD: 4°C: Four strands of DNA are left to be cooled and Taq polymerase is deactivated. | |||
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? | |||
*Adenine (A): Thymine | |||
*Thymine (T): Adenine | |||
*Cytosine (C): Guanine | |||
*Guanine (G): Cytosine | |||
Q4. During which two steps of thermal cycling does base-pairing occur? | |||
Base pairing occurs during the step of thermal cycling in which the primers are attached to the DNA template strand. It also occurs during the step in which the taq polymerase attaches the deoxyribonucleotides to the DNA template strand. Base-pairing occurs during these steps because the DNA strands are held at temperatures at which the strands are denatured but the temperature is not high enough to prevent the formation of hydrogen bonds. | |||
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Latest revision as of 23:49, 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
PCR Reaction Sample List
OpenPCR program
Denature at 95°C for 30 seconds, Anneal at 57°C for 30 seconds, and *Extend at 72°C for 30 seconds
Research and DevelopmentPCR - The Underlying Technology
Base pairing occurs during the step of thermal cycling in which the primers are attached to the DNA template strand. It also occurs during the step in which the taq polymerase attaches the deoxyribonucleotides to the DNA template strand. Base-pairing occurs during these steps because the DNA strands are held at temperatures at which the strands are denatured but the temperature is not high enough to prevent the formation of hydrogen bonds.
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