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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
- 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
|
| G9PM + |
Positive control |
none
|
| G9PM - |
Negative control |
none
|
| G9PM 1-1 |
Patient 1, replicate 1 |
52130
|
| G9PM 1-2 |
Patient 1, replicate 2 |
52130
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| G9PM 1-3 |
Patient 1, replicate 3 |
52130
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| G9PM 2-1 |
Patient 2, replicate 1 |
17921
|
| G9PM 2-2 |
Patient 2, replicate 2 |
17921
|
| G9PM 2-3 |
Patient 2, replicate 3 |
17921
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DNA Sample Set-up Procedure
- Collect supplies: Lab coat, disposable gloves, PCR reaction tubes (8 with DNA taq polymerase), Primer mix (8 with 50 micrograms each), Strip of empty PCR tubes, disposable pipette tips, micro-proprietor and PCR machine.
- Collect patient samples. Collect positive control and negative controls.
- Record patient identification numbers and properly label tubing.
- Inoculate samples with the two primers and the DNA taq PCR.
- Activate the PCR machine: Initial heating temp is 95 degrees Celsius, heating maintained for 2 minutes. DNA denatures at this temperature.
- Samples are then cooled to 57 degrees Celsius. Temperature maintained for 30 seconds. Samples are annealed at this temperature. Samples are then heated to 72 degrees Celsius to extend. Temperature maintained for 30 seconds. Step then repeated 35 times.
- Finally heat the sample to 72 degrees Celsius for 2 minutes.
- Store samples in 4 degrees Celsius.
OpenPCR program
Heated Lid: 100°C
Initial Step:95°C for two minutes
Number of Cycles: 35
Denature at 95°C for 30 seconds, Anneal at 57°C for 30 seconds, 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
Question 1:
Template DNA: The strand of DNA that will serve as the beginning structure in order to become a complete double helix in DNA synthesis.
Primers: A strand of nucleic acid that serves as a starting pint in PCR aslo reffered as base nucleotides.
Taq Polymerase: Its a thermostable DNA polymerase that is an enzyme in this process of replicating the complementary nucleotides.
Deoxyribosenucleotides(dNTP's): The monomer that is compromises into three parts which is one phosphate group, deoxyribose sugar and a nitrogenous base.
Question 2:
Initial Step:
The current research of the PCR where they are are using a template DNA sample where there is one original sample of DNA from a hair follicle, blood, skin and etc to be the start of the process.
Denature:
After setting it up in a deoxyribose nucleoside triphosphate solution mix (dNTP) in which serve as nucleotide building blocks and are essential to maintaining life, the next step is heating it up the solution to 95°C in time for denaturing the double helix into two individual strands.
Anneal:
Right after stability is achieved, the temperature is then switched to 57°C to add strands of nucleic acid or primers where they anneal to complementary matches on the two DNA strands where they bracket themselves into prepared sequences of the template DNA.
Extension:
Next comes the step of raising the temperature to 72°C and then adding the enzyme Taq Polymerase to the primed sequences.
Final Step:
The enzymes then add the remaining nucleotides to make a complete second copy. In which we have a pair of double helix DNA which are exact copies of each other
Final Hold(Repetition if necessary):
Then this first cycle can be repeated over and over again until the required amount of copied DNA is reached.
Question 3:
Base Annealing Combinations:
Adenine(A): Thymine(T)
Thymine(T): Adenine(A)
Cytosine(C): Guanine(G)
Guanine(G): Cytosine(C)
Question 4:
Base-pairing occurs during the "Anneal" and "Extend" steps of thermal cycling in which these nucleotide bases help finish creating a double helix DNA.
"Polymerase Chain Reaction | Biochemistry." Encyclopedia Britannica Online. Encyclopedia Britannica, n.d. Web. 01 Apr. 2015.
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BME 100 Spring 2015
