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OUR TEAM
LAB 4 WRITE-UP
Protocol
Materials
- Lab coat and disposable gloves
- PCR reaction mix, 8 tubes, 50 μL each: Mix contains Taq DNA polymerase, MgCl2, and dNTP’s (http://www.promega.com/resources/protocols/product-information-sheets/g/gotaqcolorless-master-mix-m714-protocol/)
- DNA/primer mix, 8 tubes, 50 μL each: Each mix contains a different template DNA. All tubes have the same forward primer and reverse primer
- A strip of empty PCR tubes
- Disposable pipette tips: only use each only once. Never re-use disposable pipette tips or samples will be cross-contaminated
- Cup for discarded tips
- Micropipettor
- OpenPCR machine: shared by two groups
PCR Reaction Sample List
| Tube Label |
PCR Reaction Sample |
Patient ID
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| G2 + |
Positive control |
none
|
| G2 - |
Negative control |
none
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| G2 1-1 |
Patient 1, replicate 1 |
15062
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| G2 1-2 |
Patient 1, replicate 2 |
15062
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| G2 1-3 |
Patient 1, replicate 3 |
15062
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| G2 2-1 |
Patient 2, replicate 1 |
95748
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| G2 2-2 |
Patient 2, replicate 2 |
95748
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| G2 2-3 |
Patient 2, replicate 3 |
95748
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DNA Sample Set-up Procedure
- 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
- 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
Research and Development
PCR - The Underlying Technology
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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BME 100 Spring 2015
