Cut the PCR tubes to size in order for them them to fit in the PCR machine.
Label each tube with the sample that will be placed inside of it.
Using the micropipette, transfer 50 microliters of the PCR reaction mix into the tube labelled as the positive control.
Transfer 50 microliters of the positive control DNA primer mixture into the same tube, using a fresh pipette tip.
Repeat steps 3 and 4 for the negative control as well as each of the patient replicate samples, using the corresponding DNA primer mixture for each tube.
Shut the lids of the PCR tubes securely.
Place the PCR tubes in the PCR machine.
Run the trial
Tube Label
PCR Reaction Sample
Patient ID
G1 +
Positive control
none
G1 -
Negative control
none
G1 1-1
Patient 1, replicate 1
78826
G1 1-2
Patient 1, replicate 2
78826
G1 1-3
Patient 1, replicate 3
78826
G1 2-1
Patient 2, replicate 1
96337
G1 2-2
Patient 2, replicate 2
96337
G1 2-3
Patient 2, replicate 3
96337
OpenPCR Program
The PCR machine works by running the sample through cycles of heating and cooling to aid in the replication of DNA through PCR. The lid of the machine is heated to 100°C. It then heats the sample to 95°C for 2 minutes. Afterwards, it cycles the sample through heating and cooling for 35 cycles. During each cycle, it denatures the sample at 95°C for 30 seconds, then anneals at 57°C for 30 seconds, and finally extends at 72°C for 30 seconds. After the cycles are completed, the PCR machine heats the sample to 72°C for 2 minutes before holding the sample at 4°C
FINAL HOLD: 4°C
Materials
Lab Coat and disposable gloves
PCR reaction mix, 8 tubes, 50 µL each
DNA/primer mix, 8 tubes, 50 µL each
A strip of empty PCR tubes
Disposable pipette tips
Cup for discarded tips
Micropipettor
OpenPCR machine
Research and Development
What is the function of each component of a PCR reaction?
The template DNA is the sample DNA that contains the target sequence. At the beginning of the reaction, high temperature is applied to the original double-stranded DNA molecule to separate the strands from each other.
Primers are short pieces of single-stranded DNA that are complementary to the target sequence. The polymerase begins synthesizing new DNA from the end of the primer.
Taq Polymerase is the enzyme that enables running the PCR at high temperature (~60°C and above), which facilitates high specificity of the primers and reduces the production of unspecific products.
Deoxyribonucleotides are single units of the bases A, T, G, and C, which are essentially "building blocks" for new DNA strands.
What happens to the components during each step of thermal cycling?
During thermal cycling, the DNA sample undergoes changes in temperature to facilitate replication. In the initial step, where the DA is heated to 95°C for 2 minutes, the DNA separates into its individual strands for replication. Afterward, the heating cycles begin. During denaturing, the sample is held at 95°C for 30 seconds to separate the DNA again into its strands. This is followed by annealing at 57°C for 30 seconds, in which DNA primers bond to the strand to prepare for transcription. Finally, the cycle ends with extension at 72°C for 30 seconds, in which taq polymerase binds to the primers and pairs the dNTP's to the base pairs on the DNA template. After all 35 cycles are completed, the sample is held at 72°C for 2 minutes to complete the process of extention. It is then cooled to 4°C before being removed from the machine.
Which base anneals to each base listed below?
Adenine anneals to Thymine
Thymine anneals to Adenine
Cytosine anneals to Guanine
Guanine anneals to Cytosine
During which two steps of thermal cycling does base-pairing occur?
Base pairing occurs during annealing and extension. During annealing, primer sequences bond with their respective sequences of base pairs on the template DNA. Afterward, during extension, taq polymerase pairs dNTP's to their complimentary bases on the template strand of DNA.
BME 100 Fall 2014
