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
Open PCR machine: shared by two groups
PCR Reaction Sample List
Tube Label
PCR Reaction Sample
Patient ID
G5 P +
Positive control
none
G5 N -
Negative control
none
G5 1-1
Patient 1, replicate 1
59697
G5 1-2
Patient 1, replicate 2
59797
G5 1-3
Patient 1, replicate 3
59797
G5 2-1
Patient 2, replicate 1
79049
G5 2-2
Patient 2, replicate 2
79049
G5 2-3
Patient 2, replicate 3
79049
DNA Sample Set-up Procedure
Gather all required materials (listed above)
Divide strip of 8 empty tubes into two sets of four
Follow the instructions from the Student Workbook and label each of the 8 tubes legibly with a black fine point marker in the following way:
G (Group)
# (Group Number)
Patient # (1 or 2) [DASH] Sample # (1, 2, or 3)
((Two of the tubes must be labelled as "control." [P or N]))
Patient Example: G5 1-2
Control Example: G5 P
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
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: This is the sequence of DNA that is intended for amplification. It will undergo extreme heat in order for it to denature.
Primers: A primer is a single strand of DNA that serves as the starting point for DNA synthesis that binds to specific DNA sequences.
Taq Polymerase: This is a thermostable enzyme that recombines nucleotides following separation by heat. It creates a new, complimentary strand of DNA through adding new nucleotides to the old ones.
Deoxyribonucleotides (dNTP's): There are the building blocks of DNA: adenine (A), thymine (T), cytosine (C) and guanine (G). These are used by the Taq Polymerase to synthesize the complementary strand of DNA.
Q2: What happens to the components (listed above) during each step of thermal cycling?
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): pairs with Thymine (T)
Thymine (T): pairs with Adenine (A)
Cytosine (C): pairs with Guanine (G)
Guanine (G): pairs with Cytosine (C)
Q4: During which two steps of thermal cycling does base-pairing occur? Explain your answers.