Start by moving the extracted DNA into a special PCR tube
Label patients extracted DNA
Next add Primer 1 to the PCR tube
Now add Primer 2
Add nucleotides to your PCR tube
Add the DNA Polymerase to the PCR tube
Place the PCR tube into a DNA thermal cycler
OpenPCR program
HEATED LID - 100°C
INITIAL STEP - 95°C for 2 minutes
NUMBER OF CYCLES - 25
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 DNA strand of interest. We want to amplify part of the DNA sequence so it is easier to view.
Primers: Attach to the part of the DNA sequence that you want to copy and amplify
Taq Polymerase: Reads and replicates the DNA sequence of interest to create more copies. Taq polymerase is used in PCR because of its unique ability to withstand high temperatures.
Deoxyribonucleotides (dNTP's): These are the building blocks of DNA that will be used to make copies of the template DNA
INITIAL STEP: 95°C for 2 minutes --> Preheats the DNA thermal cycler and establishes the desired temperature
Denature at 95°C for 30 seconds: The double stranded DNA sequence falls apart resulting in two single stranded DNA sequences
Anneal at 57°C for 30 seconds: Primers bind to their respective target sequences
Extend at 72°C for 30 seconds: At this temperature Taq polymerase is activated. Polymerase finds the primers, attaches to the DNA single strands, and begins to add complimentary nucleotides to each sequence to create the new DNA strands.
BME 100 Fall 2017
