Part 4: Polymerase Chain Reaction
We will dilute the DNA that you isolated from your salmon sample, and pipette a small
amount of the dilution into a PCR tube along with a premade "PCR master mix." The
master mix contains fish-specific COI primers to amplify part of the mitochondrial
cytochrome-c oxidase gene as well as Taq polymerase, deoxynucleotides (dNTPs), and
buffer for optimal salt & pH conditions. The DNA is diluted to reduce the chance that
contaminants (e.g., polysaccharides or proteins) not fully washed away during DNA
isolation may inhibit the reaction, which might cause the PCR reaction to fail.
Your PCR tubes will then be placed into a thermal cycler and will undergo 40 cycles of
amplification. After the PCR had completed, your tube will be held for you until next week,
where you will use gel electrophoresis to check your PCR product before sending it off for
sequencing.
WEAR GLOVES while getting up your PCR reaction!
