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Colony PCR is a convenient high-throughput method for determining the presence or absence of insert DNA in plasmid constructs. Individual transformants can either be lysed in water with a short heating step or added directly to the PCR reaction and lysed during the initial heating step.How do I set up colony PCR reactions?
The best way to do this is by using your vector with and without an insert to verify that the primers amplify the expected size PCR products. Setting up colony PCR reactions is nearly identical to preparing a standard PCR reaction: combine template, primers, polymerase, and dNTPs and then incubate with a standard PCR thermocycling program.Why is my coloncolony PCR not working?
Colony PCR can be tricky. The reason for this is the sometimes poor quality of the colony material (presence of contaminants, genomic DNA, etc.).How much DNA template do I need for a colony PCR?
PCR doesn’t need that much DNA template. Colony PCR works better from liquid cultures than from colonies from plates. This protocol can likely be optimized further. This “protocol” is an outline that you should amend with manufacturer’s instructions and internet searches.