Learn what polymerase chain reaction is, how it amplifies DNA through repeated heating and cooling cycles, and review the key reagents needed: primers, dNTPs, Taq polymerase, buffer, and template DNA.
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Design primers and gather materials
Design forward and reverse primers with similar melting temperatures that are complementary to the DNA region you want to amplify, and gather all necessary lab materials including PCR tubes, ice bucket, and the PCR machine.
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3
Prepare PCR reaction mix on ice
Place thin-walled PCR tubes on ice and prepare a 50 microliter reaction mix containing all reagents. Optionally create a master mix with common reagents like water, Taq polymerase, and dNTPs to save time on multiple reactions.
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4
Program PCR machine with thermal cycles
Set up the PCR machine with the initial denaturation step (2 minutes at 94°C) followed by 25-30 cycles of denaturation (30 seconds at 94°C), annealing (30 seconds at 5°C below primer melting temperature), and extension (1-2 minutes per kilobase at 72°C).
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Run final extension cycle
Set the PCR machine to perform a final extension for 5 minutes at 72°C to fill in any protruding ends of the newly synthesized DNA strands.
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Prepare agarose gel for analysis
While the PCR cycles are running, prepare an agarose gel for later use in verifying the PCR product.
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Verify PCR product by gel electrophoresis
After the final cycle completes, run 2 microliters of the PCR product on the agarose gel to check the size and concentration of the amplified DNA.
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Troubleshoot and optimize if needed
If the PCR reaction did not work, try adding magnesium chloride and/or DMSO to each reaction, or adjust the annealing temperature to improve specificity and product yield.