You wish to insert a gene in the plasmid below. (Note the neon green lines represent restriction sites.)
Diagram
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How could you use colony PCR to find the recombinant plasmid that has your gene of interest?
To use colony PCR to find the recombinant plasmid that has the gene of interest, the following steps can be taken:
1. Start by picking a colony from the plate containing the transformed bacteria cells.
2. Mix a small amount of the colony (around 1 μL) with a PCR mix containing forward and reverse primers that bind to the gene of interest.
3. Perform PCR amplification using the following cycling parameters: denaturation at 95°C for 5 minutes, followed by 30 cycles of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute. Finally, hold at 72°C for 5 minutes.
4. Analyze the amplification products using agarose gel electrophoresis. The presence of a band of the expected size indicates successful insertion of the gene of interest into the plasmid.
5. If the PCR product size is not as expected, pick another colony and repeat the process until the desired recombinant plasmid is identified.