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
| 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
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.
Research and Development
PCR - The Underlying Technology
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.