Colony PCR Protocol
Often times it is difficult to screen a desired recombinant plasmid for orientation, insert length, etc. The following PCR protocol in combination with the appropriate primers provides a rapid means to determine the length and orientation of a cloned insert. This protocol was introduced to FSU by Brenda Bennison after she attended a seminar at Woods Hole instructed by Chuck Wimpee of the University of Wisconsin-Milwaukee.
The original reference for this technique is: Gussow, D., and T. Clackson. 1989. Direct clone characterization from plaques and colonies by the polymerase chain reaction. Nucleic Acids Res. 17:4000. The protocol outlined below is a modification of the original method.
- Pick an isolated colony with a sterile toothpick and inoculate 50 ul of 10 mM EDTA in a microfuge tube.
- Boil 5 minutes. Vortex and then centifuge briefly.
- Use 1-2 ul of the lysate as PCR template.
Alternatively, you can set up a 5-minute soak file at 100C on the thermal cycler, followed by a 4C soak, but the lysate should be used immediately in the PCR reaction.
The lysate can be stored at -20C for subsequent use, but we have found that amplification efficiency is somewhat reduced.