LAB 4 WRITE-UP
- . 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
- OpenPCR machine: shared by two groups
PCR Reaction Sample List
| Tube Label || PCR Reaction Sample || Patient ID
| G3 + || Positive control || 91905
| G3 - || Negative control || 11553
| G3 1-1 || Patient 1, replicate 1 ||
| G3 1-2 || Patient 1, replicate 2 ||
| G3 1-3 || Patient 1, replicate 3 ||
| G3 2-1 || Patient 2, replicate 1 ||
| G3 2-2 || Patient 2, replicate 2 ||
| G3 2-3 || Patient 2, replicate 3 ||
DNA Sample Set-up Procedure
- The materials and DNA samples of the two patients were obtained along with our positive and negative control samples.
- The strip of tubes containing the PCR mixtures was cut in half so two strips of four linked tubes were created. This step was done so the tubes fit into the PCR machine.
- The sides of the PCR mixture tubes were labeled by negative and positive control, along with three individual trials of both patients. This was done using the black marker.
- The tubes were placed into a rack.
- 50 micro liters of the negative control, positive control, and each patient were transported into individual PCR mixtures. Individual pipettes were disposed after each use.
- There was a slight deviation from the guidelines, and 100 ml of patient one was placed into the second trial single PCR mixture tube. 50 ml of this was then transported into the third trial of the first patient to dilute the second and third trials.
- The PCR tubes were then closed and placed into the PCR machine.
- After closing the lid, the PCR program was started.
- 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
- FINAL STEP: 72°C for 2 minutes
- FINAL HOLD: 4°C
Research and Development
PCR - The Underlying Technology