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Frequently Asked Questions

What is the first step in colony PCR?

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.

Why don’t I use the same primers for colony PCR?

Because many cloning projects begin by PCR amplifying an “insert”, many recommend using these same primers for colony PCR. However, most convenient does not equate to best in this case. This is because the ligation reaction components contaminate your bacterial transformations and your agar plates upon plating.

What type of primers should I use to screen colonies?

The type of primers you use depends on your preferences. Either way, make sure to test your colony PCR primers before using them to screen colonies. 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.

What is colcolony PCR and how does it work?

Colony PCR is a method used to screen for plasmids containing a desired insert directly from bacterial colonies without the need for culturing or plasmid purification steps.

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