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The first and perhaps most important step to colony PCR is designing primers. There are 3 strategies for primer design: 1) insert-specific primers, 2) backbone-specific primers, and 3) orientation-specific primers. Insert-specific primers: Insert-specific primers are designed to anneal to an insert-specific sequence.What is the difference between sequencing and colony PCR?
Sequencing allows you to confirm the sequence of the insert, insert orientation, and the sequences of the junctions between the plasmid and insert DNA. Colony PCR will greatly reduce the number of clones you’ll need to send for sequencing, but won’t tell you if your products have any mutations.What are the best controls for a colony PCR?
The best controls for a colony PCR are the same ones used to verify if the colony PCR primers work in the first place: the backbone vector with and without an insert. These controls are quick references you can use when you run your PCR products out on a gel to determine if the colonies contain an insert.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.