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
DNA Sample Set-up Procedure
Label Tubes with the labels from the table above (e.g. G16 +, G16 2-2...).
Transfer the + and - controls to the PCR tubes labeled G16 + and G16 - using the micropipette.
Transfer corresponding patient 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
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
seconds
FINAL STEP: 72°C for 2 minutes
FINAL HOLD: 4°C
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.