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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
|
| G16 + |
Positive control |
none
|
| G16 - |
Negative control |
none
|
| G16 1-1 |
Patient 1, replicate 1 |
47360
|
| G16 1-2 |
Patient 1, replicate 2 |
47360
|
| G16 1-3 |
Patient 1, replicate 3 |
47360
|
| G16 2-1 |
Patient 2, replicate 1 |
31303
|
| G16 2-2 |
Patient 2, replicate 2 |
31303
|
| G16 2-3 |
Patient 2, replicate 3 |
31303
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DNA Sample Set-up Procedure
- Label Tubes with the labels from the table above (e.g. G16 +, G16 2-2...).
- Transfer 50 μL of the + and - controls to the PCR tubes labeled G16 + and G16 - using the micropipette.
- Transfer 50 μL of the corresponding patient's DNA to special PCR tube using the micropipette.
- Dispose and replace the tips between each transfer.
- Place the PCR reaction tubes in the thermal cycler.
OpenPCR program
Research and Development
PCR - The Underlying Technology
Question 1:
Question 2:
Question 3:
Question 4:
Base pairing occurs in the anneal step and final step of the thermal cycling. In the anneal step at 57°C for 30 seconds, the primer is pairing with the DNA strand. In the final step at 72°C for 3 minutes, the DNA polymerase is paring nucleotides to complete the DNA strand.
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BME 100 Spring 2015
