Colony PCR

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. This initial heating step causes the release of the plasmid DNA from the cell, so it can serve as template for the amplification reaction. Primers designed to specifically target the insert DNA can be used to determine if the construct contains the DNA fragment of interest. Alternatively, primers targeting vector DNA flanking the insert can be used to determine whether or not the insert is the correct molecular size. Insert specific primers can provide information on both the specificity and size of the insert DNA while the use of vector specific primers allows screening of multiple constructs simultaneously. Colony PCR can also be used to determine insert orientation. PCR amplification of the plasmid using an insert specific primer paired with a vector specific primer can be designed to produce an amplicon of a specific size only if the insert is in the correct orientation. In all experimental designs, presence or absence of a PCR amplicon and size of the product are determined by electrophoresis alongside a DNA size marker on an agarose gel.