The concentration of the TOL plasmid can be increased using the polymerase chain reaction (PCR). In PCR, the target DNA is mixed with two primers (in excess), a thermostable DNA polymerase, and four deoxynucleotides. The PCR process involves three cycles; denaturation, renaturation, and synthesis of DNA.
First, the reaction mixture is placed in a high temperature of about 95 oC for one minute so the double-stranded DNA molecule can denature and form two single-stranded molecules. Then the temperature is reduced to allow for DNA annealing or renaturation. In this stage, the single DNA molecule will complementary pair with the primer. Lastly, initiation of DNA synthesis occurs at 3'-OH end of each primer.
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Why must the restriction enzyme used for the digestion of TOL plasmid be inactivated immediately after the restriction experiment? How could this be achieved?