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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 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
|
| G9 + |
Positive control |
none
|
| G9 - |
Negative control |
none
|
| G9 1-1 |
Patient 1, replicate 1 |
19405
|
| G9 1-2 |
Patient 1, replicate 2 |
19405
|
| G9 1-3 |
Patient 1, replicate 3 |
19405
|
| G9 2-1 |
Patient 2, replicate 1 |
26455
|
| G9 2-2 |
Patient 2, replicate 2 |
26455
|
| G9 2-3 |
Patient 2, replicate 3 |
26455
|
DNA Sample Set-up Procedure
- 1. Label all empty tubes 1-8. The new labels will correspond with the following:
| Label |
Contents |
Patient ID
|
| Tube 1 |
Positive Control |
None
|
| Tube 2 |
Negative Control |
None
|
| Tube 3 |
1-1 |
19405
|
| Tube 4 |
1-2 |
19405
|
| Tube 5 |
1-3 |
19405
|
| Tube 6 |
2-1 |
26455
|
| Tube 7 |
2-2 |
26455
|
| Tube 8 |
2-3 |
26455
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- 2. Set the micropipettor to 50 μL. Attach a tip, and transfer the contents of the 8 PCR reaction mix tubes into the individual empty tubes. Discard the tip.
- 3. Attach a new tip to the micropipettor, set to 50 μL. Transfer the contents of the positive control DNA tube into tube #1. Discard the tip.
- 4. Attach a new tip to the micropipettor, set to 50 μL. Transfer the contents of the negative control DNA tube into tube #2. Discard the tip.
- 5. For tubes #3, #4, and #5, transfer 50 μL of the contents of the 3 DNA samples of patient 1 (ID 19405), using a new tip each time.
- 6. Repeat the process of step 5 for patient 2 (ID 26455), adding the contents to tubes #6, #7, and #8.
- 7. Make sure that each reaction tube is tightly sealed, and split the strip of eight tubes into two sets of four. Insert these sets into the OpenPCR machine, and run it as follows.
OpenPCR program
The OpenPCR program should run under certain specifics and steps. The details for these are as follows:
- The lid is heated to 100°C
- The initial step runs at 95°C for 2 minutes
- 35 cycles will be run, each with 3 steps in a cycle. The components of the cycle are denature, anneal, and extend.
- Denature runs at 95°C for 30 seconds
- Anneal runs at 57°C for 30 seconds
- Extend runs at 72°C for 30 seconds
- The final step runs at 72°C for 2 minutes
- Finally, the temperature is held at 4°C
Research and Development
PCR - The Underlying Technology
Function of Components during Thermal Cycle of a PCR Reaction
The template DNA contains the target sequence that is going to be replicated and is separated into two strands during denaturation when held at 95°C. During the annealing process, short DNA sequences known as primers complement the target sequence and provide DNA polymerase a location to synthesize new DNA when cooled to 57°C. During extension, Taq polymerase synthesizes new strands of DNA by adding deoxyribonucleotides to the Primers at a temperature of 72°C. Cooling the DNA to 4°C stops the extension cycle.
Deoxyribonucleotides
Adenine, Thymine, Cytosine, and Guanine are the four molecules that Nucleotides consist of. Pairing together using hydrogen bonds, Adenine bonds to Thymine and Cytosine bonds to Guanine, to create new strands of DNA. This base pairing occurs during the annealing and extension processes in a PCR reaction.
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BME 100 Fall 2014
