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
G2 +
Positive control
none
G2 -
Negative control
none
G2 1-1
Patient 1, replicate 1
15062
G2 1-2
Patient 1, replicate 2
15062
G2 1-3
Patient 1, replicate 3
15062
G2 2-1
Patient 2, replicate 1
95748
G2 2-2
Patient 2, replicate 2
95748
G2 2-3
Patient 2, replicate 3
95748
DNA Sample Set-up Procedure
Step 1
Step 2
Step 3...
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?
Template DNA:
Primers:
Taq Polymerase:
Deoxyribonucleotides (dNTP’s):
Q2. What happens to the components (listed above) during each step of thermal cycling?
INITIAL STEP: 95°C for 3 minutes:
*Denature at 95°C for 30 seconds:
Anneal at 57°C for 30 seconds:
Extend at 72°C for 30 seconds:
FINAL STEP: 72°C for 3 minutes:
FINAL HOLD: 4°C:
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?
Adenine (A):
Thymine (T):
Cytosine (C):
Guanine (G):
Q4. During which two steps of thermal cycling does base-pairing occur? Explain your answers.
BME 100 Spring 2015
