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.
Protocols for Colony PCR
- A-Tailing with Taq Polymerase
- PCR Protocol for Taq DNA Polymerase with Standard Taq (Mg-free) Buffer (M0320)
- PCR Protocol for Taq DNA Polymerase with ThermoPol® Buffer (M0267)
- PCR Protocol for Taq DNA Polymerase with Standard Taq Buffer (M0273)
- PCR Protocol for LongAmp® Taq DNA Polymerase (M0323)
- PCR Using Hot Start Taq DNA Polymerase (M0495)
- Robust Colony PCR from Multiple E. coli Strains using OneTaq® Quick-Load® Master Mixes
Molecular Cloning Technical Guide
The Molecular Cloning Technical Guide helps with product selection, protocols, tips for optimization and trouble-shooting.
Reagents and Tools for Molecular Cloning
Learn about recommended products for cloning in our Reagents and Tools for Molecular Cloning Brochure.
- Polymerase Fidelity: What is it, and what does it mean for your PCR?
- PCR Selector
- PCR Selection Chart
- Troubleshooting Guide for Cloning
- Guidelines for PCR Optimization with OneTaq® and OneTaq® Hot Start DNA Polymerases
Other Tools & Resources
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