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
G18 P
Positive control
none
G18 N
Negative control
none
G18 1-1
Patient 1, replicate 1
41180
G18 1-2
Patient 1, replicate 2
41180
G18 1-3
Patient 1, replicate 3
41180
G18 2-1
Patient 2, replicate 1
68784
G18 2-2
Patient 2, replicate 2
68784
G18 2-3
Patient 2, replicate 3
68784
DNA Sample Set-up Procedure
Collect a small sample of DNA from cells such as blood, skin, saliva or even hair follicles
Move your extracted DNA sample into a special PCR tube
Add primer 1 to the PCR tube (Primer 1 attaches to a site on the DNA strand at either end of the template segment)
Add primer 2 (which attaches to the 2nd site of the template segment on the DNA strand) to the PCR tube
Add nucleotides to the PCR tube
Add DNA polymerase to the PCR tube
Now place the PCR tube containing all of the reaction components into the DNA thermal cycler
Push start button on DNA 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
Q1. What is the function of each component of a PCR reaction?
Q2. What happens to the components (listed above) during each step of thermal cooling?
Q3. DNA is made up of four types of molecules called nucleotides, designated as A, T, C and G. Base-pairing, driven by hydrogen bonding, allows base pairs to stick together. Which base anneals to each base listed below?